1-(1- (2-Ethoxyethyl)-3-Ethyl-7-(4-Methylpyridin-2-Ylamino) - 1H-Pyrazolo [4,3-D] Pyrimidin-5-YL) Piperidine-4-Carboxylic acid and salts thereof

ABSTRACT

The present invention comprises 1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylic acid and its salts. The invention further comprises pharmaceutical compositions, methods of treatment, and synthetic methods relating to 1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylic acid and its salts.

FIELD OF THE INVENTION

The present invention relates generally to1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid and its salts. The present invention further relates topharmaceutical compositions comprising this compound or its salts,methods of treatment employing this compound or its salts, and methodsof preparing this compound or its salts. In general, the compound andits salts inhibit the cyclic guanylate monophosphate-specificphosphodiesterase type 5 (“PDE5”) enzyme.

BACKGROUND OF THE INVENTION

Hypertension is a condition associated with, among other physiologicalproblems, an increased risk of stroke, myocardial infarction, atrialfibrillation, heart failure, peripheral vascular disease and renalimpairment. Despite the numerous drugs available in variouspharmacological categories to treat hypertension and relatedphysiological problems, not all patients respond to such drugs aseffectively or as safely as desired. Additional therapeutic agents forthe treatment of hypertension and/or related conditions are stillneeded.

One class of therapeutic agents reported in the literature as useful forthe treatment of hypertension are inhibitors of the PDE5 enzyme (“PDE5inhibitors”). In general, vascular endothelial cells secrete nitricoxide which acts on vascular smooth muscle cells and leads to theactivation of guanylate cyclase and the accumulation of cyclic guanosinemonophosphate (“cGMP”). The accumulation of cGMP causes the muscles torelax and the blood vessels to dilate, leading to a reduction in bloodpressure. The cGMP is inactivated by hydrolysis to guanosine5′-monophosphate (“GMP”) by cGMP-specific phosphodiesterases. OnecGMP-phosphodiesterase involved in the inactivation of cGMP is the PDE5enzyme. Inhibitors of the PDE5 enzyme decrease the rate of cGMPhydrolysis. This reduction in cGMP hydrolysis potentiates the actions ofnitric oxide leading to a lowering of blood pressure.

Compounds that are PDE5 inhibitors have been reported in the literature.For example, WO2005049616 reports one class ofpyrazolo[4,3-d]pyrimidinyl compounds. WO2005049617 reports another classof pyrazolo[4,3-d]pyrimidinyl compounds. WO2004096810 reports anotherclass of pyrazolo[4,3-d]pyrimidinyl compounds. EP 1348707 reportsanother class of pyrazolo[4,3-d]pyrimidinyl compounds.

The identification of additional compounds that are PDE5 inhibitors isdesirable. Such compounds can be used to treat subjects suffering fromor susceptible to hypertension and/or related physiological problems andfurther expand the range of treatment options available for suchsubjects.

SUMMARY OF THE INVENTION

In one embodiment, the invention comprises1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid and its pharmaceutically acceptable salts.

In another embodiment, the invention comprises a pharmaceuticalcomposition comprising1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier.

In another embodiment, the invention comprises a pharmaceuticalcomposition comprising1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof, one or moreadditional therapeutic agents, and a pharmaceutically acceptablecarrier.

In another embodiment, the invention comprises methods for treating acondition in a subject by administering to a subject a therapeuticallyeffective amount of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof. Conditions that canbe treated in accordance with the present invention includecardiovascular conditions, metabolic conditions, central nervous systemconditions, pulmonary conditions, sexual dysfunction, pain, and renaldysfunction.

In another embodiment, the invention comprises methods for treating acondition in a subject by administering to a subject a therapeuticallyeffective amount of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof, one or moreadditional therapeutic agents, and a pharmaceutically acceptablecarrier. Conditions that can be treated in accordance with the presentinvention include cardiovascular conditions, metabolic conditions,central nervous system conditions, pulmonary conditions, sexualdysfunction, pain, and renal dysfunction.

In another embodiment, the invention comprises use of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof, for the manufactureof a medicament for the treatment of a condition in a subject. Suchconditions include cardiovascular conditions, metabolic conditions,central nervous system conditions, pulmonary conditions, sexualdysfunction, pain, and renal dysfunction.

In another embodiment, the invention comprises methods for making1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid, or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph illustrating the effect on blood pressure ofrepeated oral administration of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid (1 mg/kg daily oral dose), alone and in combination with enalapril,in a conscious spontaneous hypertensive rat model.

FIG. 2 shows a graph illustrating the effect on 24-hour urinary cGMP ofrepeated oral administration of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid (1 mg/kg daily oral dose), alone and in combination with enalapril,in a conscious spontaneous hypertensive rat model.

DETAILED DESCRIPTION OF THE INVENTION

This detailed description of embodiments is intended only to acquaintothers skilled in the art with applicants' inventions, its principles,and its practical application so that others skilled in the art mayadapt and apply the inventions in their numerous forms, as they may bebest suited to the requirements of a particular use. These inventions,therefore, are not limited to the embodiments described in thisspecification, and may be variously modified.

A. ABBREVIATIONS AND DEFINITIONS

As used in reference to ¹H NMR, the symbol “δ” refers to a ¹H NMRchemical shift.

As used in reference to ¹H NMR, the abbreviation “br” refers to a broad¹H NMR signal.

As used in reference to ¹H NMR, the abbreviation “d” refers to a doublet¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “m” refers to amultiplet ¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “q” refers to a quartet¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “s” refers to a singlet¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “t” refers to a triplet¹H NMR peak.

The abbreviation “BSA” refers to bovine serum albumin.

The abbreviation “CDI” refers to carbodiimide.

The abbreviation “DMSO” refers to dimethylsulfoxide.

The abbreviation “DBAD” refers to dibenzylazodicarboxylate.

The abbreviation “EDTA” refers to ethylenediaminetetraacetic acid.

The abbreviation “HEPES” refers to4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

The abbreviation “HRMS” refers to High Resolution Mass Spectroscopy(electrospray ionisation positive scan).

The abbreviation “iPrOAc” refers to isopropyl acetate.

The abbreviation “LCMS” refers to Liquid Chromatography MassSpectroscopy.

The abbreviation “m/z” refers to a Mass spectrum peak.

The abbreviation “NaN(TMS)₂” refers to sodium hexamethyldisilazide.

The abbreviation “pfu” refers to plaque-forming units.

The abbreviation “Pt/C” refers to platinum on carbon.

The abbreviation “PMSF” refers to phenylmethylsulfonyl fluoride.

The abbreviation “SPA” refers to scintillation proximity assay.

The abbreviation “THF” refers to tetrahydrofuran.

The abbreviation “Tris-HCl” refers to Tris(hydroxymethyl)aminomethanehydrochloride.

The term “cGMP-mediated condition” refers to any condition mediated bycGMP, whether through direct regulation by cGMP, or through indirectregulation by cGMP as a component of a signalling pathway.

The term “PDE5-mediated condition” refers to any condition mediated bythe PDE5 enzyme.

The term “hypertensive subject” refers to a subject having hypertension,suffering from the effects of hypertension or susceptible to ahypertensive condition if not treated to prevent or control suchhypertension.

The term “pharmaceutically acceptable carrier” refers to a carrier thatis compatible with the other ingredients of the composition and is notdeleterious to the subject. Such carriers may be a pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting a chemical agent. The preferred compositiondepends on the method of administration.

The term “therapeutically effective amount” refers to that amount ofdrug or pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system or animal that is being sought by aresearcher or clinician.

The term “treatment” (and corresponding terms “treat” and “treating”)includes palliative, restorative, and preventative treatment of asubject. The term “palliative treatment” refers to treatment that easesor reduces the effect or intensity of a condition in a subject withoutcuring the condition. The term “preventative treatment” (and thecorresponding term “prophylactic treatment”) refers to either preventingthe onset of a preclinically evident condition altogether or preventingthe onset of a preclinical evident stage of a condition in a subject.The term “restorative treatment” refers to treatment that halts theprogression of, reduces the pathologic manifestations of, or entirelyeliminates a condition in a subject.

B. CARBOXYPIPERIDINE COMPOUND

In one embodiment, the present invention comprises the compound havingthe structure:

and the salts (particularly the pharmaceutically-acceptable salts) ofthe compound. The corresponding name of this compound is1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid. Unless otherwise stated, this compound, all tautomeric forms ofthe compound, and the pharmaceutically-acceptable salts of the compoundand its tautomeric forms are collectively referred to in thisapplication as the “Carboxypiperidine Compound”. The CarboxypiperidineCompound is useful, for example, as an inhibitor of the PDE5 enzyme.

In one embodiment, the present invention is directed to the free acidform of the Carboxypiperidine Compound.

In another embodiment, the present invention is directed to thepharmaceutically acceptable salts of the Carboxypiperidine Compound.

WO2004096810 reports a genus of compounds that generically embraces theCarboxypiperidine Compound. Although WO2004096810 provides examples ofspecific compounds within the genus, it does not disclose theCarboxypiperidine Compound itself. The Carboxypiperidine Compound,however, possesses at least one or more different properties relative tothe specific compounds described in WO2004096810. These propertiesinclude, for example, efficacy (e.g, greater in vitro, ex vivo, and/orin vivo potency), safety (e.g., greater selectivity and/or lowertoxicity), pharmacokinetic properties (e.g., C_(max), longer half-lifeand/or lower clearance), and manufacturing properties (e.g., ease ofsynthesis and/or availability of starting materials).

C. SALTS

As noted above, the Carboxypiperidine Compound may be in either the freeacid form or in a salt form. Different salt forms of theCarboxypiperidine Compound may have different physical propertiesrelative to each other. Accordingly, selection of the specific salt formof the Carboxypiperidine Compound potentially can impact, for example,compound stability (such as over a range of temperatures and/orhumidities), compound solubility, and other compound physical propertiesthat can affect a drug product. In addition, salts of theCarboxypiperidine Compound generally will have greater aqueoussolubility than the corresponding free acid form.

Where the salt of the Carboxypiperidine Compound is administered to ahuman or animal subject (as opposed to, for example, use for in vitrotesting), the salt preferably is a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” refers to a salt that isgenerally considered suitable for human consumption (particularly anon-toxic salt). Pharmaceutically acceptable salts include base additionsalts and acid addition salts of the corresponding free acid. Thesesalts typically may be prepared by conventional means from the free acidof the Carboxypiperidine Compound.

Illustrative base addition salts of the Carboxypiperidine Compoundinclude metallic salts and organic salts. Metallic salts include alkalimetal (group Ia) salts (such as lithium, sodium and potassium salts),alkaline earth metal (group IIa) salts (such as calcium and magnesiumsalts), and other physiological acceptable metal salts (such as aluminumand zinc salts). Organic salts include salts made from secondary,tertiary and quaternary amines (such as tromethamine, diethylamine,triethylamine, ethanolamine, diethanolamine, triethanolamine,ethylenediamine, N,N′-dibenzylethylenediamine, meglumine(N-methylglucamine), procaine, chloroprocaine, and choline) and saltsmade from cationic amino acids (such as arginine, lysine and histidine).

Examples of suitable acid addition salts include, hydrochloride,hydrobromide, hydrofluoride, hydroiodide, borate, fluoroborate,phosphate, hydrogen phosphate, dihydrogen phosphate, glycerophosphate,hexafluorophosphate, metaphosphate, nitrate, bicarbonate, carbonate,bisulphate, sulfate, dodecylsulfate, sulfonate, methanesulfonate,ethanesulfonate, benzenesulfonate, toluenesulfonate,trifluoromethanesulfonate, toluenesulfonate, 2-hydroxyethanesulfonate,cyclohexylaminosulfonate, 2-naphthalesulfonate, camphorsulfonate,acetate, adipate, anthranilate, aspartate, ascorbate, algenate,trifluoroacetate, phenylacetate, benzoate, p-hydroxybenzoate, besylate,butyrate, β-hydroxybutyrate, camphorate, camsyate, citrate, embonate,edisylate, esylate, formate, fumarate, gluconate, digluconate,glycolate, glucamate, glucuronate, gluceptate, heptanoate,glycoheptanoate, hexanoate, hibenzate, isethionate, isothionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, nicotinate,orotate, oxalate, palmitate, pamoate, panthothenate, pectinate, picrate,pivalate, propionate, cyclopentanepropionate, 3-phenylpropionate,pyruvate, saccharate, salicylate, stearate, succinate, sulfanilate,tartrate, galactarate, tosylate, uronate, galacturonate, thiocyanate,and undecanoate.

For a review on suitable salts, see “Handbook of Pharmaceutical Salts:Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH,Weinheim, Germany, 2002).

D. TAUTOMERS

As used in this application, the term Carboxypiperidine Compound (aswell as the corresponding structure) is intended to embrace alltautomeric isomers of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicacid. Representative tautomeric isomers of the CarboxypiperidineCompound are shown below:

E. METHODS OF TREATMENT

The present invention further comprises methods for treating a conditionin a subject having or susceptible to having such a condition byadministering to the subject a therapeutically-effective amount of theCarboxypiperidine Compound. In one embodiment, the treatment ispreventative treatment. In another embodiment, the treatment ispalliative treatment. In still another embodiment, the treatment isrestorative treatment.

In another embodiment, the condition is a PDE5-mediated condition.

In another embodiment, the condition is a cGMP-mediated condition. Acondition in which, for instance, insufficient cGMP is a majorcomponent, and whose production or action is modulated in response tothe PDE5 enzyme, would therefore be considered a disorder mediated bycGMP.

In another embodiment, the condition is selected from the groupconsisting of cardiovascular conditions, metabolic conditions, centralnervous system conditions, pulmonary conditions, sexual dysfunction,pain and renal dysfunction.

In another embodiment, the condition is a cardiovascular conditionselected from the group consisting of hypertension (including essentialhypertension, pulmonary hypertension, pulmonary arterial hypertension,secondary hypertension, isolated systolic hypertension, hypertensionassociated with diabetes, hypertension associated with atherosclerosis,and renovascular hypertension); complications associated withhypertension (including vascular organ damage, congestive heart failure,angina, stroke, glaucoma and impaired renal function); valvularinsufficiency; stable, unstable and variant (Prinzmetal) angina;peripheral vascular disease; myocardial infarct; stroke (includingstroke recovery); thromboembolic disease; restenosis; arteriosclerosis;atherosclerosis; angiostenosis after bypass; angioplasty (includingpercutaneous transluminal angioplasty and percutaneous transluminalcoronary angioplasty); hyperlipidemia; hypoxic vasoconstriction;vasculitis (including Kawasaki's syndrome); heart failure (includingcongestive heart failure, decompensated heart failure, systolic heartfailure, diastolic heart failure, left ventricular heart failure, rightventricular heart failure, and left ventricular hypertrophy); Raynaud'sphenomenon; preeclampsia; pregnancy-induced high blood pressure;cardiomyopathy; and arterial occlusive disorders.

In another embodiment, the condition is hypertension.

In another embodiment, the condition is pulmonary hypertension.

In another embodiment, the condition is pulmonary arterial hypertension.

In another embodiment, the condition is heart failure.

In another embodiment, the condition is diastolic heart failure.

In another embodiment, the condition is systolic heart failure.

In another embodiment, the condition is angina.

In another embodiment, the condition is thrombosis.

In another embodiment, the condition is stroke (including strokerecovery).

In another embodiment, the condition is a condition associated withendothelial dysfunction (including conditions selected from the groupconsisting of atherosclerotic lesions, myocardial ischaemia, peripheralischaemia, valvular insufficiency, pulmonary arterial hypertension,angina, clots, vascular complications after vascular bypass, vasculardilation, vascular repermeabilisation, and heart transplantation).

In another embodiment, the condition is a metabolic condition selectedfrom the group consisting of Syndrome X; diabetes (including type I andtype II diabetes); insulin resistance; syndromes of insulin resistance(including insulin receptor disorders, Rabson-Mendenhall syndrome,leprechaunism, Kobberling-Dunnigan syndrome, Seip syndrome, Lawrencesyndrome, Cushing syndrome, acromegaly, pheochromocytoma, glucagonoma,primary aldosteronism, somatostatinoma, Lipoatrophic diabetes, β-celltoxin induced diabetes, Grave's disease, Hashimoto's thyroiditis andidiopathic Addison's disease); impaired glucose tolerance; diabeticcomplications (including diabetic gangrene, diabetic arthropathy,diabetic nephropathy, diabetic glomerulosclerosis, diabeticderamatopathy, diabetic neuropathy, peripheral diabetic neuropathy,diabetic cataract, and diabetic retinopathy); hyperglycemia; andobesity.

In another embodiment, the condition is insulin resistance.

In another embodiment, the condition is nephropathy.

In another embodiment, the condition is a central nervous systemcondition selected from the group consisting of vascular dementia;craniocerebral trauma; cerebral infarct; cerebrovascular accident,dementia; concentration disorders; Alzheimer's disease; Parkinson'sdisease; amyolateral sclerosis; Huntington's disease; multiplesclerosis; Creutzfeld-Jacob disease; anxiety; depression; sleepdisorders; and migraine.

In another embodiment, the condition is Alzheimer's disease.

In another embodiment, the condition is Parkinson's disease.

In another embodiment, the condition is amyolateral sclerosis.

In another embodiment, the condition is a concentration disorder.

In another embodiment, the condition is a pulmonary condition selectedfrom the group consisting of asthma; acute respiratory distress; cysticfibrosis; chronic obstructive pulmonary disease; bronchitis; and chronicreversible pulmonary obstruction.

In another embodiment, the condition is pain. In another embodiment, thecondition is acute pain. Examples of acute pain include acute painassociated with injury or surgery. In another embodiment, the conditionis chronic pain. Examples of chronic pain include neuropathic pain(including postherpetic neuralgia and pain associated with peripheral,cancer or diabetic neuropathy), carpal tunnel syndrome, back pain(including pain associated with herniated or ruptured intervertabraldiscs or abnormalities of the lumber facet joints, sacroiliac joints,paraspinal muscles or the posterior longitudinal ligament), headache,cancer pain (including tumour related pain such as bone pain, headache,facial pain or visceral pain) or pain associated with cancer therapy(including postchemotherapy syndrome, chronic postsurgical painsyndrome, post radiation syndrome, pain associated with immunotherapy,or pain associated with hormonal therapy), arthritic pain (includingosteoarthritis and rheumatoid arthritis pain), chronic post-surgicalpain, post herpetic neuralgia, trigeminal neuralgia, HIV neuropathy,phantom limb pain, central post-stroke pain and pain associated withchronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinalcord injury, Parkinson's disease, epilepsy and vitamin deficiency. Inanother embodiment, the condition is nociceptive pain (including painfrom central nervous system trauma, strains/sprains, burns, myocardialinfarction and acute pancreatitis, post-operative pain (pain followingany type of surgical procedure), posttraumatic pain, renal colic, cancerpain and back pain). In another embodiment, the condition is painassociated with inflammation (including arthritic pain (such asosteoarthritis and rheumatoid disease pain), ankylosing spondylitis,visceral pain (including inflammatory bowel disease, functional boweldisorder, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome,functional abdominal pain syndrome, Crohn's disease, ileitis, ulcerativecolitis, dysmenorrheal, cystitis, pancreatitis and pelvic pain). Inanother embodiment, the condition is pain resulting frommusculo-skeletal disorders (including myalgia, fibromyalgia,spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articularrheumatism, dystrophinopathy, glycogenolysis, polymyositis andpyomyositis). In another embodiment, the condition is selected from thegroup consisting of heart and vascular pain (including pain caused byangina, myocardical infarction, mitral stenosis, pericarditis, Raynaud'sphenomenon, scleredoma and skeletal muscle ischemia). In anotherembodiment, the condition is selected from the group consisting of headpain (including migraine such as migraine with aura and migraine withoutaura), cluster headache, tension-type headache mixed headache andheadache associated with vascular disorders; orofacial pain, includingdental pain, otic pain, burning mouth syndrome and temporomandibularmyofascial pain).

In another embodiment, the condition is sexual dysfunction (includingsexual dysfunction selected from the group consisting of impotence(organic or psychic); male erectile dysfunction; clitoral dysfunction;sexual dysfunction after spinal cord injury; female sexual arousaldisorder; female sexual orgasmic dysfunction; female sexual paindisorder; and female hypoactive sexual desire disorder).

In another embodiment, the condition is male erectile dysfunction.

In another embodiment, the condition is renal dysfunction (includingrenal dysfunction selected from the group consisting of acute renalfailure, chronic renal failure; nephropathy (such as diabeticnephropathy); tubulointerstitial disorders; glomerulopathy; andnephritis. In another embodiment, the condition is a cancer conditionselected from the group consisting of cancerous cachexia; tumormetastasis and neoplasia.

In another embodiment, the condition is osteoporosis.

In another embodiment, the condition is a gastrointestinal conditionselected from the group consisting of nutcracker oesophagus; analfissure; disorders of gut motility; irritable bowel syndrome andhaemorrhoids. In another embodiment, the condition is a urologiccondition selected from the group consisting of bladder outletobstruction; incontinence and benign prostatic hyperplasia.

In another embodiment, the condition is a skin condition, selected frompsoriasis; urticaria and skin necrosis.

In another embodiment, the condition is an ophthalmic condition selectedfrom retinal disease; macular degeneration and glaucoma.

In another embodiment, the condition is nitrate intolerance.

In another embodiment, the condition is baldness.

In another embodiment, the condition is a gynecologic condition selectedfrom the group consisting of dysmenorrhoea (primary and secondary);infertility and premature labor. In another embodiment, the condition issecondary dysmenorrhoea.

In another embodiment, the present invention further comprises methodsfor inducing weight loss or maintenance of weight loss in a subject byadministering to the subject a therapeutically-effective amount of theCarboxypiperidine Compound.

F. SUBJECTS

The Carboxypiperidine Compound (including the corresponding methods oftreatment and pharmaceutical compositions) are suitable for use with,for example, mammalian subjects such as humans, other primates (e.g.,monkeys, chimpanzees), companion animals (e.g., dogs, cats, horses),farm animals (e.g., goats, sheep, pigs, cattle), laboratory animals(e.g., mice, rats), and wild and zoo animals (e.g., wolves, bears,deer). In one embodiment, the subject is a mammalian subject. In anotherembodiment, the subject is a human.

G. HYPOTHESIZED MECHANISM

Without being held to a particular theory, it is hypothesized that theCarboxypiperidine Compound is an inhibitor of the PDE5 enzyme. It isfurther hypothesized that the Carboxypiperidine Compound inhibits theaction of the PDE5 enzyme leading to an increase in intracellular cGMPlevels. This increase in intracellular cGMP levels reduces intracellularcalcium signaling, which in turn results in vascular smooth musclerelaxation and a reduction in blood pressure.

H. ADMINISTRATION AND DOSING

The Carboxypiperidine Compound is generally administered in atherapeutically effective amount. In one embodiment, theCarboxypiperidine Compound is administered in a PDE5 enzyme inhibitingamount. The Carboxypiperidine Compound can be administered by anysuitable route in the form of a pharmaceutical composition adapted tosuch a route, and in a dose effective for the treatment intended.Therapeutically effective doses of the Carboxypiperidine Compoundrequired to prevent or arrest the progress of, or to treat the medicalcondition, are readily ascertained by one of ordinary skill in the artusing preclinical and clinical approaches familiar to the medicinalarts.

The dosage regimen for the compounds and/or compositions containing thecompounds is based on a variety of factors, including the type, age,weight, sex and medical condition of the patient; the severity of thecondition; the route of administration; and the activity of theparticular compound employed. Thus the dosage regimen may vary based onthe specific situation. Dosage levels from about 0.001 mg to about 100mg of Carboxypiperidine Compound per kilogram of body weight per day areuseful in the treatment of the above-indicated conditions. In oneembodiment, the total daily dose of the Carboxypiperidine Compound(administered in single or divided doses) is typically from about 0.001mg/kg to about 20 mg/kg (i.e., mg compound/kg body weight). In anotherembodiment, the total daily dose of the Carboxypiperidine Compound isfrom about 0.005 mg/kg to about 10 mg/kg. In another embodiment, thetotal daily dose is from about 0.005 mg/kg to about 5 mg/kg. In anotherembodiment, the total daily dose is from about 0.01 mg/kg to about 1mg/kg. These dosages are based on an average human subject having aweight of about 65 kg to about 75 kg. A physician will readily be ableto determine doses for subjects whose weight falls outside of thisrange, such as infants. The administration of the CarboxypiperidineCompound can be repeated a plurality of times in a day (typically nogreater than 4 times) to achieve the desired daily dose.

For convenience the Carboxypiperidine Compound can be administered in aunit dosage form. If desired, multiple doses per day of the unit dosageform can be used to increase the total daily dose. The unit dosage form,for example, may be a tablet or capsule containing about 0.01, 0.05,0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 250 or 500 mg of theCarboxypiperidine Compound. In one embodiment, the unit dosage formcontains from about 0.01 mg to about 500 mg of the CarboxypiperidineCompound. In another embodiment, the unit dosage form contains fromabout 0.05 mg to about 250 mg of the Carboxypiperidine Compound. Inanother embodiment, the unit dosage form contains from about 0.1 mg toabout 100 mg of the Carboxypiperidine Compound. In another embodiment,the unit dosage form contains from about 0.5 mg to about 50 mg of theCarboxypiperidine Compound.

I. USE IN THE PREPARATION OF A MEDICAMENT

In another embodiment, the present invention comprises theCarboxypiperidine Compound for use as a medicament (such as a unitdosage tablet or unit dosage capsule).

The present invention further comprises the use of the CarboxypiperidineCompound for the manufacture of a medicament (such as a unit dosagetablet or unit dosage capsule) to treat one or more of the conditionspreviously identified in the above sections discussing methods oftreatment. In one embodiment, the condition is hypertension.

J. PHARMACEUTICAL COMPOSITIONS

For the treatment of the conditions referred to above, theCarboxypiperidine Compound can be administered as the free acid compoundper se. In another embodiment, the Carboxypiperidine Compound can beadministered as one or more pharmaceutically acceptable salts of thefree acid compound per se. In another embodiment, the CarboxypiperidineCompound can be administered as a mixture of the free acid compound perse and one or more pharmaceutically acceptable salts of the free acidcompound per se.

The present invention further comprises pharmaceutical compositionscomprising the Carboxypiperidine Compound. In one embodiment, thepharmaceutical composition comprises the Carboxypiperidine Compound inthe free acid form. In another embodiment, the pharmaceuticalcomposition comprises one or more pharmaceutically acceptable salts ofthe Carboxypiperidine Compound. In another embodiment, thepharmaceutical composition comprises a mixture of the CarboxypiperidineCompound in the free acid form and one or more pharmaceuticallyacceptable salts of the Carboxypiperidine Compound. In one embodiment,the pharmaceutical composition comprises the Carboxypiperidine Compoundand at least one pharmaceutically-acceptable carrier. The carrier can bea solid, a liquid, or both, and may be formulated with theCarboxypiperidine Compound as a unit dosage form, for example, a tablet,which can contain from 0.05% to 95% by weight of the CarboxypiperidineCompound. Other pharmacologically active substances can also be present.

The Carboxypiperidine Compound may be administered by any suitableroute, preferably in the form of a pharmaceutical composition adapted tosuch a route, and in a dose effective for the treatment intended. TheCarboxypiperidine Compound and corresponding compositions, for example,may be administered orally, rectally, parenterally, or topically.

Oral administration of a solid dosage form may be, for example,presented in discrete units, such as hard or soft capsules, pills,cachets, lozenges, or tablets, each containing a predetermined amount ofat least one compound of the present invention. In another embodiment,the oral administration may be in a powder or granule form. In anotherembodiment, the oral dose form is sub-lingual, such as, for example, alozenge. In such solid dosage forms, the Carboxypiperidine Compound isordinarily combined with one or more adjuvants. In the case of capsules,tablets, and pills, the dosage forms also may comprise buffering agentsor may be prepared with enteric coatings.

In another embodiment, oral administration may be in a liquid dose form.Liquid dosage forms for oral administration include, for example,pharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs containing inert diluents commonly used in the art (e.g.,water). Such compositions also may comprise adjuvants, such as wetting,emulsifying, suspending, flavoring (e.g., sweetening), and/or perfumingagents.

In another embodiment, the present invention comprises a parenteral doseform. “Parenteral administration” includes, for example, subcutaneousinjections, intravenous injections, intraperitoneally, intramuscularinjections, intrasternal injections, and infusion. Injectablepreparations (e.g., sterile injectable aqueous or oleaginoussuspensions) may be formulated according to the known art using suitabledispersing, wetting agents, and/or suspending agents.

In another embodiment, the present invention comprises a topical dosageform. “Topical administration” includes, for example, transdermaladministration, such as via transdermal patches or iontophoresisdevices, intraocular administration, or intranasal or inhalationadministration. Compositions for topical administration also include,for example, topical gels, sprays, ointments, and creams. A topicalformulation may include a compound which enhances absorption orpenetration of the active ingredient through the skin or other affectedareas. When the compounds of this invention are administered by atransdermal device, administration will be accomplished using a patcheither of the reservoir and porous membrane type or of a solid matrixvariety. Formulations suitable for topical administration to the eyeinclude, for example, eye drops wherein the compound of this inventionis dissolved or suspended in suitable carrier. For intranasaladministration or administration by inhalation, the active compounds ofthe invention are conveniently delivered in the form of a solution orsuspension from a pump spray container that is squeezed or pumped by thepatient or as an aerosol spray presentation from a pressurized containeror a nebulizer, with the use of a suitable propellant.

In another embodiment, the present invention comprises a rectal dosageform. Such rectal dose form may be in the form of, for example, asuppository.

Other carrier materials and modes of administration known in thepharmaceutical art may also be used. Pharmaceutical compositions of theinvention may be prepared by any of the well-known techniques ofpharmacy, such as effective formulation and administration procedures.The above considerations in regard to effective formulations andadministration procedures are well known in the art and are described instandard textbooks. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds.,Handbook of Pharmaceutical Excipients (3^(rd) Ed.), AmericanPharmaceutical Association, Washington, 1999.

K. COMBINATIONS AND COMBINATION THERAPY

The Carboxypiperidine Compound also can be administered in combinationwith other therapeutic agents to treat the various conditions previouslydiscussed above. The Carboxypiperidine Compound and the othertherapeutic agent(s) may be may be administered simultaneously (eitherin the same dosage form or in separate dosage forms) or sequentially.Accordingly, in one embodiment, the present invention comprises methodsfor treating a condition in a subject having or susceptible to havingsuch a condition by administering to the subject atherapeutically-effective amount of the Carboxypiperidine Compound andone or more additional therapeutic agents. In another embodiment, thepresent invention comprises a pharmaceutical composition comprising theCarboxypiperidine Compound, one or more additional therapeutic agents,and a pharmaceutically acceptable carrier.

In one embodiment, the Carboxypiperidine Compound may be administeredwith aspirin.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more diuretics. Examples of suitablediuretics include hydroclorothiazide (such as MICROZIDE™ and ORETIC™),hydroflumethiazide (such as SALURON™), bemetanide (such as BUMEX™),torsemide (such as DEMADEX™), metolazone (such as ZAROXOLYN™),chlorothiazide (such as DIURIL™, ESIDRIX™ and HYDRODIURIL™), triamterene(such as DYRENIUM™), ethacrynic acid (such as EDECRIN™), chlorthalidone(such as HYGROTON™), furosemide (such as LASIX™), indapamide (such asLOZOL™), and amiloride (such as MIDAMOR™ and MODURETIC™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more angiotensin converting enzymeinhibitors. Examples of suitable angiotensin converting enzymeinhibitors include quinapril (such as ACCUPRIL™), perindopril (such asACEON™), captopril (such as CAPOTEN™), enalapril (such as VASOTEC™),ENALAPRILAT™, ramipril (such as ALTACE™), cilazapril, delapril,fosenopril (such as MONOPRIL™), zofenopril, indolapril, benazepril (suchas LOTENSIN™), lisinopril (such as PRINIVIL™ and ZESTRIL™), spirapril,trandolapril (such as MAVIK™), perindep, pentopril, moexipril (such asUNIVASC™) fasidotril, S-allymercaptocaptopril, and pivopril.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more angiotensin II receptor blockers.Examples of suitable angiotensin II receptor blockers includecandesartan (such as ATACAND™), eprosartan (such as TEVETEN™),irbesartan (such as AVEPRO™), losartan (such as COZAAR™), olmesartan,olmesartan medoxomil (such as BENICAR™), tasosartan, telmisartan (suchas MICARDIS™), valsartan (such as DIOVAN™), zolasartan, FI-6828K,RNH-6270, UR-7198, Way-126227, KRH-594, TAK-536, BRA-657, and TA-606.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more calcium channel blockers. Examples ofsuitable calcium channel blockers include nifedipine (such as ADALAT™,ADALAT CC™ and PROCARDIA™), verapamil (such as CALAN™, COVERA-HS™,ISOPTIN SR™ and VERELAN™), diltiazem (such as CARDIZEM™ CARDIZEM CD™,CARDIZEM LA™, CARDIZEM SR™, DILACOR™, TIAMATE™ and TIAZAC™), isradipine(such as DYNACIRCT™ and DYNACIRC CR™), amlodipine (such as NORVASC™),felodipine (such as PLENDIL™), nisoldipine (such as SULAR™), bepridil(such as VASCOR™), vatanidipine, clevidipine, lercanidipine, dilitiazem,and NNC-55-0396.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more beta blockers. Examples of suitablebeta blockers include timolol (such as BLOCARDEN™), carteolol (such asCARTROL™), carvedilol (such as COREG™), nadolol (such as CORGARD™),propranolol (such as INNOPRAN XL™), betaxolol (such as KERLONE™),penbutolol (such as LEVATOL™), metoprolol (such as LOPRESSOR™ andTOPROL-XL™), atenolol (such as TENORMIN™), pindolol (such as VISKEN™),and bisoprolol.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more alpha blockers. Examples of suitablealpha blockers include prazosin, doxazosin (such as CARDURA™),phenoxybenzamine (such as DIBENZYLINE™), terazosin (such as HYTRIN™),CDRI-93/478 and CR-2991.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more alpha-beta blockers. An example of asuitable alpha-beta blocker is labetalol (such as NORMODYNE™ orTRANDATE™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more aldosterone receptor antagonists.Examples of suitable aldosterone receptor antagonists include eplerenone(such as INSPRA™) and spironolactone (such as ALDACTONE™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more renin inhibitors. Examples of suitablerenin inhibitors include aliskiren (SPP 100), SPP-500/600 and YS-004-39.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more central antiadrenergics. Examples ofsuitable central antiadrenergics includes methyldopa (such as ALDOMET™),clonidine (such as CATAPRES™ or CATAPRES-TTS™), guanfacine (such asTENEX™), and guanabenz (such as WYTENSIN™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more glycosides/inotropic agents. An exampleof a suitable glycoside/inotropic agent is digoxin (such as LANOXIN™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more human B-type natriuretic peptides. Anexample of a suitable human B-type natriuretic peptide is nesiritide(such as NATRECOR™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more organic nitrates or nitric oxidedonors. “Nitric oxide donor” refers to a compound that donates, releasesand/or directly or indirectly transfers a nitrogen monoxide species,and/or stimulate the endogenous production of nitric oxide orendothelium-derived relaxing factor (EDRF) in vivo and/or elevateendogenous levels of nitric oxide or EDRF in vivo. It also includescompounds that are substrates for nitric oxide synthase. Examples ofsuitable nitric oxide donors include S-nitrosothiols, nitrites,nitrates, N-oxo-N-nitrosamines, SPM 3672, SPM 5185, SPM 5186 andanalogues thereof, sodium nitroprusside, nitroglycerin, isosorbidedinitrate, isosorbide mononitrate, molsidomine, SIN-1 and substrates ofthe various isozymes of nitric oxide synthase.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more bradykinin agonists.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more soluble guanylate cyclase activators.An example of a suitable soluble guanylate cyclase activator isBAY-41-8543.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more neutral endopeptidase inhibitors.Examples of suitable neutral endopeptidase inhibitors includeomapatrilat, fasidotril, mixanpril, sampatrilat, Z13752A,

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more endothelian antagonists. Examples ofsuitable endothelin antagonists include ambrisentan, darusentan,J-104132, SPP-301, TBC-3711, YM-62899, YM-91746 and BMS-193884.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more 3-hydroxy-3-methylglutaryl coenzyme Areductase inhibitors. Examples of suitable 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors include fluvastatin (such as LESCOL™),atorvastatin (such as LIPITOR™), lovastatin (such as ALTOCOR™ orMEVACOR™), pravastatin (such as PRAVACHOL™), rosuvastatin (such asCRESTOR™), and simvastatin (such as ZOCOR™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with niacin or one or more nicotinic acid derivatives.Examples of suitable niacin or nicotinic acid derivatives includeNIACOR™, NIASPAN™, NICOLAR™, and SLO-NIACIN™.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more fibric acid derivatives. Examples ofsuitable fibric acid derivatives include clofibrate (such asATROMID-S™), gemfibrozil (such as LOPID™), and fenofibrate (such asTRICOR™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more bile acid sequestants. Examples ofsuitable bile acid sequestants include colestipol (such as COLESTID™),cholestyramine (such as LOCHOLEST™, PREVALITE™, QUESTRAN™, and QUESTRANLIGHT™), colesevelam (such as WELCHOL™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more cholesterol absorption inhibitors. Anexample of a suitable cholesterol absorption inhibitor is ezetimibe(such as ZETIA™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more cholesteryl ester transport proteininhibitors. An example of a suitable cholesteryl ester transport proteininhibitor is torcetrapib.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more apical sodium-dependent bile acidcotransporter inhibitors. Examples of suitable apical sodium-dependentbile acid cotransporter inhibitors include SD-5613, AZD7806 and 264W94.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more alpha glucosidase inhibitors. Examplesof suitable alpha glucosidase inhibitors include miglitol (such asGLYSET™) and acarbose (such as PRECOSE™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more biguanides. Examples of suitablebiguanides include rosiglitazone (such as AVANDAMET™) and metformin(such as GLUCOPHAGE™ and GLUCOPHAGE XR™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more insulins. Examples of suitable insulinsinclude HUMALOG™, HUMALOG 50/50™, HUMALOG 75/25™, HUMULIN 50/50™,HUMALIN 75/25™, HUMALIN L™, HUMALIN N™, HUMALIN R™, HUMALIN R U-500™,HUMALIN U™, ILETIN II LENTE™, ILETIN II NPH™, ILETIN II REGULAR™LANTUS™, NOVOLIN 70/30™, NOVILIN N™, NOVILIN R™, NOVOLOG™, VELOSULINBR™, and EXUBERA™.

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more meglitnides. Examples of suitablemeglitnides include repaglinide (such as PRANDIN™) and nateglinide (suchas STARLIX™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more sulfonylureas. Examples of suitablesulfonylureas include glimepiride (such as AMARYL™), glyburide (such asDIABETA™, GLYNASE PRESTAB™ or MICRONASE™), and glipizide (such asGLUCOTROL™ and GLUCOTROL XL™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more thiazolidinediones. Examples ofsuitable thiazolidinediones include pioglitazone (such as ACTOS™) androsiglitazone (such as AVANDIA™).

In another embodiment, the Carboxypiperidine Compound may beco-administered with one or more alpha-2-delta ligands. Examples ofsuitable alpha-2-delta ligands include gabapentin, pregabalin (such asLYRICA™), [(1R,5R,6S)-6-(aminomethyl)bicyclo[3.2.0]hept-6-yl]aceticacid, 3-(1-aminomethyl-cyclohexylmethyl)-4H-[1,2,4]oxadiazol-5-one,C-[1-(1H-tetrazol-5-ylmethyl)-cycloheptyl]-methylamine,(3S,4S)-(1-aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid,(1α,3α,5α)-(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid,(3S,5R)-3-aminomethyl-5-methyl-octanoic acid,(3S,5R)-3-amino-5-methyl-heptanoic acid,(3S,5R)-3-amino-5-methyl-nonanoic acid and(3S,5R)-3-amino-5-methyl-octanoic acid),(2S,4S)-4-(3-Chlorophenoxy)praline, and(2S,4S)-4-(3-Fluorobenzyl)praline.

L. KITS

The present invention further comprises kits that are suitable for usein performing the methods of treatment described above. In oneembodiment, the kit comprises a first dosage form comprising theCarboxypiperidine Compound and a container for the first dosage form. Inanother embodiment, the kit comprises a first dosage form comprising theCarboxypiperidine Compound and a second dosage form comprising a secondtherapeutic agent.

In another embodiment, the kit comprises a first dosage form comprisingthe Carboxypiperidine Compound and a second dosage form comprising anangiotensin converting enzyme inhibitor.

In another embodiment, the kit comprises a first dosage form comprisingthe Carboxypiperidine Compound and a second dosage form comprising anangiotensin II receptor antagonist.

In another embodiment, the kit comprises a first dosage form comprisingthe Carboxypiperidine Compound and a second dosage form comprising analdosterone receptor antagonist.

In another embodiment, the kit comprises a first dosage form comprisingthe Carboxypiperidine Compound and a second dosage form comprising anitric oxide donor.

M. COMPOUND SYNTHESIS

The Carboxypiperidine Compound may be prepared using the methodsillustrated in the synthetic schemes and the experimental proceduresdescribed below. The general synthetic schemes are presented forpurposes of illustration and are not intended to be limiting. Thestarting materials used to prepare the Carboxypiperidine Compound arecommercially available or may prepared by routine methods well known tothose of ordinary skill in the art (such as those methods disclosed instandard reference books such as the COMPENDIUM OF ORGANIC SYNTHETICMETHODS, Vol. I-VI (published by Wiley-Interscience)).

The following examples illustrate the synthesis of the free acid of theCarboxypiperidine Compound:

Example 1

1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicAcid Step 1: Preparation of1-(2-ethoxyethyl)-3-ethyl-4-nitro-1H-pyrazole-5-carboxamide

A 1 L flask was charged with 3-ethyl-4-nitropyrazole-5-carboxamide(prepared as described in EP 1176142 at page 18) (15.0 g, 81 mmol),triphenylphosphine (25.6 g, 97.8 mmol), and tetrahydrofuran (120 mL).The resulting mixture was cooled by placing the flask in a 0° C. bathand 2-ethoxyethanol (9.5 ml, 98 mmol) was added to the flask. A solutionof di-tert-butyl azodicarboxylate (22.5 g, 97.8 mmol) in tetrahydrofuran(90 mL) was then added to the flask over a period of 2.5 hours. Themixture was stirred with cooling for an additional hour and then warmedto room temperature and stirred for one additional hour. The mixture wasagain cooled to 0° C., treated with 6M hydrochloric acid (40 ml), andheated to 40° C. for one hour. The mixture was partially concentratedand extracted between ethyl acetate and water. The aqueous layer wasback-extracted with ethyl acetate. The combined organic layers werewashed with water, washed with brine, dried over magnesium sulfate,filtered and then concentrated. The resulting viscous oil was treatedwith 2-propanol and this mixture was then concentrated. The resultingmaterial was treated with 2-propanol and heated. Upon cooling, theresulting crystals were filtered, rinsed with cold 2-propanol, and driedto afford 16.5 g of the title compound.

Step 2: Preparation of4-amino-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazole-5-carboxamide

1-(2-ethoxyethyl)-3-ethyl-4-nitro-1H-pyrazole-5-carboxamide from step 1(15.0 g, 58 mmol), 5% Platinum-on-carbon (0.75 g), and isopropyl acetate(150 mL) were combined and the mixture was stirred under 80 psi ofhydrogen for about 50 hours. The mixture was then filtered throughcelite, the celite was rinsed with acetonitrile, and the filtrate wasconcentrated. The material eventually crystallized to give 13.7 g as ahydrate of the title compound.

Step 3:1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione

A mixture of carbonyldiimidazole (12.3 g, 76.1 mmol) in acetonitrile(123 mL) was warmed to 50° C. The4-amino-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazole-5-carboxamide prepared instep 3 (13.2 g, 58 mmol) was added to the mixture over a period of onehour and the temperature of the mixture was increased to 75° C. duringthe addition. The mixture was then cooled and stirred overnight. Themixture was cooled in an ice bath and filtered. The filter cake wasrinsed with water and dried to provide 12.8 g of the title compound.

Step 4: Preparation of5,7-dichloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine

A mixture of1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dionefrom step 3 (25.2 g, 100 mmol) was treated with PhPOCl₂ (195 g, 1000mmol) and heated, under N2, with stirring at 135° C. for 20 h. Themixture was cooled and then heated at 140° C. for an additional 20 h.The mixture was cooled and slowly added to an ice (600 g)/water (200 mL)mixture. This was stirred for 3 h and the resulting solid was collectedby filtration and rinsed with water (2×100 mL). The solid was suspendedin ethyl acetate (300 mL). Water (200 mL) was added and the pH adjustedto 1-2. Then 10% NaHCO₃ (125 mL) was added (resulting in pH ˜7) and thelayers were separated. The organic layer was successively washed withwater (400 mL) and saturated sodium chloride solution (150 mL). Theorganic solution was then dried, and concentrated to provide 23 g of thetitle compound.

Step 5: Preparation ofN-[5-chloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-4-methylpyridin-2-ylamine

A mixture of 2-amino-4-picoline (4.32 g, 40 mmol) and5,7-dichloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine fromstep 4 (5.78 g, 20 mmol) was treated with THF (25 mL) and cooled to 0°C. The mixture was stirred and treated with 1 M LiN(TMS)₂ in THF (40 mL,40 mmol) at a rate such that the temperature was kept below 5° C. Themixture was stirred for 30 min and was then treated with 10% citric acidsolution until pH 6-7 was achieved. The mixture was partiallyconcentrated under reduced pressure. The mixture was then stirred at 5°C. for 1 h. The resulting solid was collected by suction filtration andwashed with water (40 mL). The solid was dried in a vacuum oven at atemperature less than 60° C. to afford 7.0 g of the title compound.

Step 6: Preparation of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicAcid

A mixture ofN-[5-chloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-4-methylpyridin-2-ylaminefrom step 5 (5.42 g, 166 mmol), isonipecotic acid (85.9 g, 665 mmol),cesium carbonate (162 g, 498 mmol), and DMSO (55 mL) was heated, withstirring, at 125° C. After 20 h, the mixture was cooled to 20° C. andwater (165 mL) was added. The mixture was stirred for 15 min and wasthen treated with ethyl acetate (55 mL). The layers were separated andthe aqueous was treated with another portion of ethyl acetate (55 mL).The layers were separated again and the aqueous layer was taken to pH ˜5using 6 M HCl. After stirring for 1 hour, the resulting solid wasfiltered, washed with water (20 mL) and dried in vacuuo, to give 5.5 gof the title compound. ¹H NMR (400 MHz, DMSO-d₆):

1.10 (t, 3H), 1.30 (t, 3H), 1.49-1.60 (m, 2H), 1.84-1.95 (m, 2H),2.32-2.36 (m, 3H), 2.52-2.57 (m, 1H), 2.78 (q, 2H), 2.99-3.08 (m, 2H),3.52 (q, 2H), 3.78 (t, 2H), 4.45-4.53 (m, 2H), 4.56-4.64 (m, 2H), 6.91(d, 1H), 8.05 (s, 1 H), 8.18 (d, 1H), 9.63 (s, 1H), 12.18 (s, 1H). LCMSm/z 454.

Alternatively, a mixture ofN-[5-chloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-4-methylpyridin-2-ylaminefrom step 5 can be combined with isonipecotic acid (between 1 to 10equivalents) and a base and heated between 100-125° C. in a solventuntil the reaction is complete. Suitable bases include cesium carbonate,sodium carbonate and potassium carbonate. Suitable solvents include DMSOand N,N-dimethylformamide. Upon cooling, water can be added and thebasic solution extracted (between 1 to 3 times) with an organic solventsuch as ethyl acetate. The remaining basic layer is acidified to pH 5with HCl. The mixture is stirred for approximately 1 hour. The solid isfiltered off, washed with water, and dried in vaccuo to provide thetitle compound.

Example 2

1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicAcid Step 1: Preparation of1-(2-ethoxyethyl)-3-ethyl-4-nitro-1H-pyrazole-5-carboxamide

1-(2-ethoxyethyl)-3-ethyl-4-nitro-1H-pyrazole-5-carboxamide was preparedaccording to step 1, Example 1 Step 2: Preparation of4-amino-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazole-5-carboxamide

A mixture of 1-(2-ethoxyethyl)-3-ethyl-4-nitro-1H-pyrazole-5-carboxamidefrom step 1 (5.70 g, 22 mmol) and palladium hydroxide (1.0 g) in ethanol(110 mL) was treated with ammonium formate (7.01 g, 111 mmol) in fourunequal portions at about 10 minute intervals. The resulting mixture washeated under reflux for two hours and then cooled. The mixture wasfiltered through celite and concentrated. The resulting residue waspurified by column chromatography on silica gel using a mixture of ethylacetate and hexane as the eluent to provide 2.45 g of the titlecompound. ¹H NMR (400 MHz, CDCl₃): δ1.17 (t, 3H), 1.21-1.32 (m, 3 H),2.60 (q, 2H), 3.53 (q, 2H), 3.79-3.95 (m, 2H), 4.38-4.52 (m, 2H). MS(ESI) m/z 227.

Step 3:1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione

A mixture of 4-amino-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazole-5-carboxamidefrom step 2 (2.44 g, 10.8 mmol) in N,N-dimethylformamide (40 mL) wastreated with carbonyldiimidazole (1.92 g, 11.9 mmol) and heated at 80°C. overnight. The mixture was concentrated in vacuuo and the residue waspurified by column chromatography on silica gel using a mixture of ethylacetate and hexane as the eluent to provide 1.04 g of the titlecompound. MS (ESI) m/z 253.

Step 4: Preparation of5,7-dichloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine

A mixture of the1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dioneprepared in step 3 (7.2 g) and phosphorus oxychloride (200 mL) with onedrop of N,N-dimethylformamide was heated at reflux overnight. Themixture was cooled and the phosphorus oxychloride was evaporated underreduced pressure. Ice was added to the resulting residue and then themixture was extracted with chloroform. The organic layers were driedover magnesium sulfate, filtered, and evaporated to provide 2.09 g of5,7-dichloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidine asan oil which solidified upon standing. LCMS m/z 289.

Step 5: Preparation of1-(1-(2-ethoxyethyl)-3-ethyl-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)piperidine-4-carboxylicAcid

The 5,7-dichloro-1-(2-ethoxyethyl)-3-ethyl-1H-pyrazolo[4,3-d]pyrimidineprepared in step 4 (1 mmol), 2-amino-4-picoline (2 mmol),N,N-diisopropylethylamine (3 mmol), and N-methylpyrrolidone (1 mL) wereadded to each of two reaction vessels. Each vessel was irradiated in aCEM Discover microwave for 15 minutes at 150° C. Isonipecotic acid (3mmol) was added to each reaction vessel and the resulting mixtureirradiated for 15 minutes at 180° C. The contents of the two reactionvessels were combined. Water and ethyl acetate was added to the mixtureand the mixture was shaken. Hydrochloric acid (1M) was added to themixture and the mixture was shaken again. The layers were separated. Theorganic layer was washed with water, hydrochloric acid (1M) was added,and the layers were separated. The organic layer was dried overmagnesium sulfate, filtered, and evaporated. The pH of the aqueous layerwas raised to about six by adding saturated sodium carbonate and theaqueous layer was then extracted with ethyl acetate. The organic layerwas dried over magnesium sulfate, filtered, added to the above organicresidue and evaporated. The solution was purified by RP-HPLC on a VarianDynamax C-18 column (250×41.4 mm) with a gradient of 10-95%acetonitrile/water (hydrochloric acid) over 15 minutes in two batches.Fractions were combined and evaporated to afford 0.26 g of the titlecompound. ¹H NMR (400 MHz, DMSO-d₆):

1.10 (t, 3H), 1.30 (t, 3H), 1.49-1.60 (m, 2H), 1.84-1.95 (m, 2H),2.32-2.36 (m, 3H), 2.52-2.57 (m, 1H), 2.78 (q, 2H), 2.99-3.08 (m, 2H),3.52 (q, 2H), 3.78 (t, 2H), 4.45-4.53 (m, 2H), 4.56-4.64 (m, 2H), 6.91(d, 1H), 8.05 (s, 1H), 8.18 (d, 1H), 9.63 (s, 1H), 12.18 (s, 1H). LCMSm/z 454.

Protocols N. POTENCY AND SELECTIVITY ASSAYS Method 1: Human PlateletPDE5 Enzyme Inhibition Scintillation Proximity Assay

An in vitro assay can be used to evaluate the inhibition of PDE5 enzymeactivity by a test compound. As described more specifically below, thisassay measures the PDE50 value for the test compound (i.e., theconcentration of the test compound required to inhibit by 50% the PDE5enzyme-catalyzed hydrolysis of cGMP to GMP relative to the activity ofuninhibited controls).

The PDE5 enzyme for use in the assay can be obtained from humanplatelets by appropriate modification of the method of Thompson, W J etal.; Biochemistry 18(23), 5228-5237, 1979, as described by Ballard S Aet al.; J. Urology 159(6), 2164-2171, 1998. The PDE5 enzyme isolatedfrom human platelets can be used to catalyze the hydrolysis of [³H]cGMP(Amersham Biosciences) to 5′ nucleotide [³H]GMP. The [³H]GMP binds toyttrium silicate SPA beads (Amersham Biosciences) and is detected byscintillation counting. More specifically, the effect of the testcompound at different concentrations can be evaluated in the assay bycontacting the compound with a fixed amount of PDE5 enzyme in thepresence of substrate (cGMP or cAMP in a 3:1 ratio unlabelled to[³H]-labeled). Scintillation counting can be used as described above todetermine relative PDE5 enzyme activity. The inhibition of PDE5 enzymeactivity is then calculated relative to total PDE5 enzyme activity ofuninhibited controls.

PDE5 IC₅₀ Assay: 96-Well Microtiter Plate Format Reagents Buffer A: 20mM Tris-HCl, 5 mM MgCl₂, pH 7.4

Buffer B: 2 mg/ml BSA in Buffer A (enzyme buffer)cGMP substrate: Final concentration of 500 nM in assayThe amount of ³H-labeled substrate added depends upon the specificactivity of [³H]cGMP, and the cGMP substrate is diluted with a 10 mMstock of cold cGMP in Buffer A for a final substrate concentration of500 nM in the assay.PDE enzyme: Prepared in Buffer B. The dilution factor is determined byenzyme activity.SPA beads: 20 mg/ml suspension prepared in dH2O.

Positive Control Negative Control Standard/Test compound  2 μl 100% DMSO 2 μl 100% DMSO  2 μl Standard/Test compound 25 μl Buffer A 25 μl BufferA 25 μl Buffer A 25 μl Enzyme 25 μl Buffer B 25 μl Enzyme 50 μlSubstrate 50 μl Substrate 50 μl Substrate 50 μl SPA to stop 50 μl SPA tostop 50 μl SPA to stop

Stocks of standard and test compounds are prepared at 5 mM in 100% DMSO.The compound is serially diluted in a dilution plate using a 10-point ½log dilution format. 2 μl of the compound dilution is added in duplicateto the wells of the assay plate. 2 μl of 100% DMSO are added todesignated control wells. 25 μl of Buffer A are added to all wells. 25μl of Buffer B are added to the negative control wells. 25 μl of enzymeare added to the remaining wells. 50 μl of substrate are added to eachwell. Plates are sealed and incubated for 60 minutes on a plate shakerat 30 C. 50 μl of SPA beads are added to stop the reaction. The platesare again sealed and shaken for 15 minutes to allow the beads to bindthe GMP product. The beads are allowed to settle for 30 minutes and thenread on a NXT TopCount scintillation counter. Data are analyzed with acurve fitting application for plate-based screening. Percent inhibitionin this assay is calculated as follows:

Inhibition(%)=[(mean maximum−compound value/(mean maximum−meanminimum)]×100.

The IC₅₀ value is determined from sigmoid dose-response curves of enzymeactivity versus compound concentration.

The Carboxypiperidine Compound was tested in accordance with Method 1.The corresponding PDE5 IC₅₀ values measured are reported in Table A.

TABLE A METHOD 1: RUN NO. PDE5 IC₅₀(nM) 1 0.050 2 0.044 AVERAGE: 0.047

Compounds previously disclosed in examples from WO2004096810 were testedin accordance with Method 1. The corresponding PDE5 IC₅₀ values measuredare reported in Table B.

TABLE B METHOD 1: WO2004096810 PDE5 IC₅₀ EXAMPLE NO. STRUCTURE (nM) 1

16.2 2

0.48 3

43.8 4

8.84 7

6.03 8

6.95 9

508 11

0.29 12

1.74 13

19.1 14

42.1 15

0.09 16

0.98 17

22.4 18

25.8 19

0.49 21

2.04 23

0.39 24

37.7 26

1.83 28

47.9 34

50.2 35

737 36

1450 37

>10,000 38

>10,000 39

456 40

334 71

152 72

177 77

>10,000 80

1940 81

118 83

134 84

247 88

>100 90

1550 91

22.6 92

26.5 93

31.1 94

43.2 95

76.0 96

87.9 98

231 99

239 100

67.2 103

76.6 108

778 109

27.3 110

513 113

1250 114

60.6 115

220 116

1760 123

248 124

78.2 125

182 126

2230 127

7580 128

1390 129

139 130

>10,000 133

4.45 134

6.57 135

0.92 136

1.09 137

1.43 138

0.33 139

0.55 140

1.95 141

0.15 142

22.7 143

81.2 144

5.34 145

7.15 146

32.1 147

2.46 148

4.39 149

34.8 150

1.83 151

22.0 152

12.1 153

19.5 154

12.6 155

13.4 156

1.80 157

16.7 158

0.71 159

16.3 160

7.22 161

0.50 162

0.24 164

3.46 165

10.5 166

11.5 167

2.95 168

0.81 170

2100 173

396 174

432 176

274 177

535 179

18.3 180

341 181

0.39 182

4.03 185

1.30 188

0.47 191

0.24 192

3.06 193

1.01 194

0.56 195

0.96 196

0.56 197

1.76 198

10.0 199

3.13 200

4.70 201

25.9 202

5.46 203

0.63 204

24.1 205

6.24 206

6.09 207

1.55 208

0.71 210

82.7 211

1.57 212

0.13 213

0.42 214

0.37 215

5.30 216

60.9 217

2.55 218

11.5 219

41.0 220

3.60 221

35.4 222

10.1 225

1.33 226

0.83 227

0.35 228

3.09 229

1.61 231

10.3 233

3.85 234

8.79 235

>100 236

2.51 237

0.67 240

>100 241

48.7 242

0.62 244

2.54 245

1.55 246

4.46 248

0.30 249

0.16 250

2.37 251

0.25 252

2.81 253

1.20 254

0.59 255

1.43 256

3.82 258

1.99 259

3.74 261

0.57 262

0.93 263

0.27

Method 1A: Alternative Human Platelet PDE5 Enzyme InhibitionScintillation Proximity Assay

The PDE5 IC₅₀ of a test compound also can be measured in an alternativein vitro assay that varies from Method 1 as described below:

PDE5 IC₅₀ Assay 96-Well Microtiter Plate Format Reagents Buffer A: 20 mMTris-HCl, 5 mM MgCl₂, pH 7.4

Buffer B: 2 mg/ml BSA in Buffer A (enzyme buffer)cGMP substrate: Final concentration of 50 nM in assayThe amount of ³H-labeled substrate added depends upon the specificactivity of [³H]cGMP, and it is diluted in Buffer A.PDE enzyme: Prepared in Buffer B. The dilution factor is determined byenzyme activity.SPA beads: 4 mg/ml suspension prepared in dH₂O.

Positive Control Negative Control Standard/Test compound  3 μl 100% DMSO 3 μl 100% DMSO  3 μl Standard/Test compound 27 μl Buffer A 27 μl BufferA 27 μl Buffer A 30 μl Enzyme 30 μl Buffer B 30 μl Enzyme 30 μlSubstrate 30 μl Substrate 30 μl Substrate 30 μl SPA to stop 30 μl SPA tostop 30 μl SPA to stop

Stocks of standard and test compound are prepared at 2 mM in 100% DMSO.The test compound is serially diluted in a dilution plate using a10-point ⅕ log dilution format such that the starting concentration inthe assay is 2 μM for an initial IC₅₀ screen; confirmatory IC₅₀s aredone using a 10-point ⅓ log dilution. 27 μl of Buffer A are added to thewells of the assay plates. From the dilution plate, 3 μl of dilutedcompound is delivered in duplicate or 3 μl of 100% DMSO (for positiveand negative controls) are added. 30 μl of enzyme are added. For thenegative control wells, Buffer B is substituted in place of the enzyme.30 μl of labeled substrate are added to all wells.

After incubating for 60 minutes at room temperature, the reaction isstopped with the addition of 30 μl of the yttrium silicate beads. Thesebeads are dense and require constant agitation while being added to theplate. The plates are sealed and shaken on a plate shaker for fifteenminutes to allow the beads to bind the GMP product.

After allowing the beads to settle for 30 minutes, plates are read on aNXT TopCount scintillation counter and the data are analyzed as follows.Percent inhibition values are calculated using the means of the 0% and100% controls on each plate. The estimates of the 4-parameters of thelogistic, sigmoid dose-response model are then calculated using thewell-level percent inhibition value for the compound. The formula forthe four-parameter logistic model may be expressed asY=((a−d)/(1+(X/c)̂b))+d, where Y is the response, X is the concentration,a is the lower asymptote (minimum response), d is the upper asymptote(maximum response), c is the model IC₅₀ (in the same units as X), and bis the slope (as described in De Lean, A., P. J. Munson, and D. Rodbard(“Simultaneous analysis of families of sigmoidal curves: application tobioassay, radioligand assay, and physiological dose-response curves.”Am. J. Physiol. 235(2): E97-E102, 1978). These estimates are used tocalculate the concentration that corresponds to 50% inhibition.

The Carboxypiperidine Compound was tested in accordance with Method 1a.The corresponding PDE5 IC₅₀ values measured are reported in Table C.

TABLE C METHOD 1A: RUN NO. PDE5 IC₅₀(nM) 1 0.051 2 0.051 3 0.039 4 0.0455 0.023 6 0.026 AVERAGE: 0.039

Compounds previously disclosed in examples from WO2004096810 were testedin accordance with Method 1a. The corresponding PDE5 IC₅₀ valuesmeasured are reported in Table D.

TABLE D WO2004096810 METHOD 1a: EXAMPLE NO. PDE5 IC₅₀(nM) 261 0.115 2620.0755 263 0.0197

Method 2: Human Retina PDE6 Enzyme Inhibition Scintillation ProximityAssay (SPA)

The inhibition of PDE6 enzyme activity by a test compound can bemeasured in accordance with the in vitro assay of Method 1, but insteadusing semi-purified PDE6 enzyme isolated from human, bovine or canineretina in place of PDE5 enzyme. This assay measures the PDE6 IC₅₀ valuefor the test compound (i.e., the concentration of the test compoundrequired to inhibit by 50% the PDE6 enzyme-catalyzed hydrolysis of[³H]cGMP to the 5′ nucleotide [³H]GMP relative to the activity ofuninhibited controls). [3H]GMP binds to yttrium silicate SPA beads andis detected by scintillation counting.

PDE6 Purification: PDE6 can be isolated and purified from the following:Soluble rod PDE6/Rod Outer Segment PDE6 from Bovine eyes (Charles RiverLtd, France); Cone PDE6 from Canine eyes (LAS); or Soluble rod and ConePDE6 from Human eyes (I.I.A.M.).

Homogenisation Buffer 20 mM Hepes (Sigma) 2.383 gms 1 mM EDTA (Sigma)0.186 gms

Dissolve in 300 ml of HPLC (Acros) or double deionised water.100 mM PMSF (Sigma) 0.174 gms/10 mls ethanolAdd 5 ml of PMSF solution to buffer slowly to avoid precipitation.

Add 250 mM Sucrose (Fisher) 42.78 gms

Adjust volume to 500 ml, stir until dissolved.Adjust pH to 7.2 using 1M NaOH.

Store at 4° C.

Preparation of the Retina: Allow the eyes to thaw until soft and untilthe cornea and lens are clear. While supporting the eye with the corneauppermost, make a small incision through the conjunctiva below the levelof the iris. The incision should be sufficient to allow the humor toescape through the cut. Using fine scissors, cut around the cornea toenable it to be lifted off as a flap. Using a small hook, engage thelens and gently pull it out of orbit, taking care not to pull out theretina at the same time. Allow the retina to drop down of its own accordto the base of the orbit.

Lift the retina with care and locate the optic nerve. Cut through theoptic nerve and transfer the retina to a small dish containing a few mlof homogenisation buffer. Gently float the retina out on the surface ofthe buffer and remove any obvious pigmented material from the iris.Place the retina in a second dish with clean buffer to remove lasttraces of pigment and then transfer to a 50 ml Corning Costar centrifugetube (Sigma) and keep on ice.

PDE6 Isolation and Purification from Retina: Allow 2 mls of thehomogenisation buffer per bovine retina and 1 ml per retina for dog andhuman. Homogenise using hand-held homogeniser 3×5 second bursts coolingon ice between each burst. Filter homogenate through two layers ofsurgical gauze. Spin at 100,000 g for 60 minutes at 4° C. Filter cytosoleither through a 0.22 μM Steril-D pack if there is sufficient volume orthrough a 0.22 μM syringe-end filter (e.g., Millex®-GV). Run throughFPLC (Pharmacia FPLC LKB/FRAC-100, Pharmacia) or aliquot and store inliquid nitrogen as needed.

Method 2A: Alternative Human Retina PDE6 Enzyme Inhibition ScintillationProximity Assay (SPA)

The inhibition of PDE6 enzyme activity by a test compound also can bemeasured in accordance with the in vitro assay of Method 1A, but insteadusing semi-purified PDE6 enzyme isolated from human, bovine or canineretina in place of PDE5 enzyme. This assay measures the PDE6 IC₅₀ valuefor the test compound (i.e., the concentration of the test compoundrequired to inhibit by 50% the PDE6 enzyme-catalyzed hydrolysis of[³H]cGMP to the 5′ nucleotide [³H]GMP relative to the activity ofuninhibited controls). [3H]GMP binds to yttrium silicate SPA beads andis detected by scintillation counting.

Method 3: Human Recombinant PDE11 Enzyme Inhibition ScintillationProximity Assay (SPA)

The inhibition of PDE11 enzyme activity by a test compound can bemeasured in accordance with the in vitro assay of Method 1, but insteadusing PDE11 enzyme expressed in Sf9 insect cells in place of PDE5enzyme. This assay measures the PDE11 IC₅₀ value for the test compound(i.e., the concentration of the test compound required to inhibit by 50%the PDE11 enzyme-catalyzed hydrolysis of [³H]cGMP to the 5′ nucleotide[³H]GMP relative to the activity of uninhibited controls). [3H]GMP bindsto yttrium silicate SPA beads and is detected by scintillation counting.

PDE11 Expression and Purification:

(a) Expression: Infect 200 ml of Sf9 insect cells (Invitrogen, Carlsbad,Calif.) (1×10⁶/ml) with a multiplicity of infection of 2 using abaculovirus expression vector containing transposed PDE-11A1 (SEQ IDNO: 1) (pFastBac system, Invitrogen) (4×10⁷ pfu/ml). Incubate at 27° C.with 220 rpm shaking for 48 hours. Harvest the infected cells bypelleting at 200 rpm for 10 minutes at 4° C. Store the infected cellpellet at −80° C. until ready for purification. Resuspend the thawedcell pellets to 1×10⁷ cells/ml (=20 ml buffer consisting of 20 mM Hepes(pH7.2), 1 mM EDTA, 20 mM sucrose, 150 mM NaCl and protease inhibitortablets). Mix thoroughly and allow to stand on ice for 10 minutes.Sonicate for 5×5 seconds, on ice. Spin the mixture at 12,000 rpm for 10minutes at 4° C. in SS34. Store the insoluble pellet at −80° C., andproceed to the purification of the soluble material.(b) Purification: Prepare the column (1.0 cm diameter, Kontes) byrinsing the empty column twice with TBS. Then mix beads (Anti-FLAG M2Agarose, Sigma) with TBS and add to the column—allow the beads tosettle. Wash the column with 3 column volumes of 0.1M glycine (pH3.5).Then wash the column with 5 column volumes of TBS (do not allow to rundry). Apply the extract to the column and allow the extract to enter bygravity flow. Reapply the flow-through. Wash the column with 15 columnvolumes of TBS. Elute the protein with 5×1 ml aliquots of FLAG peptide(Sigma) (i.e., 100 ug/ml in TBS). Elute the remaining protein with 1 ml0.1M glycine, and immediately add 25 ul 1M Tris pH8 to neutralize.

Method 3A: Alternative Human Recombinant PDE11 Enzyme InhibitionScintillation Proximity Assay (SPA)

The inhibition of PDE11 enzyme activity by a test compound also can bemeasured in accordance with the in vitro assay of Method 1A, but insteadusing PDE11 enzyme expressed in Sf9 insect cells in place of PDE5enzyme. This assay measures the PDE11 IC₅₀ value for the test compound(i.e., the concentration of the test compound required to inhibit by 50%the PDE11 enzyme-catalyzed hydrolysis of [³H]cGMP to the 5′ nucleotide[³H]GMP relative to the activity of uninhibited controls). [3H]GMP bindsto yttrium silicate SPA beads and is detected by scintillation counting.

The Carboxypiperidine Compound was tested in accordance with Method 2,Method 2A, Method 3, and Method 3A. The corresponding PDE6 and PDE11IC₅₀ values measured are reported in Table E.

TABLE E METHOD METHOD METHOD METHOD 2: PDE6 2A: PDE6 3: PDE11 3A: PDE11RUN NO. IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) 1 4.01 5.12 19.5 14.3 23.89 2.43 20.0 16.5 3 4.88 16.8 4 2.12 26.0 5 5.74 28.4 6 3.72 19.6AVERAGE: 3.95 3.74 19.8 19.7

Compounds previously disclosed in examples from WO2004096810 were testedin accordance with Method 2, Method 2A, Method 3, and Method 3A. Thecorresponding PDE6 and PDE11 IC₅₀ values measured are reported in TableF.

TABLE F METHOD METHOD METHOD METHOD WO2004096810 2: PDE6 2a: PDE6 3:PDE11 3a: PDE11 EXAMPLE NO. IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) 245250 390 246 657 4320 248 9.92 316 249 5.10 1190 250 221 3120 251 9.511862 252 307 874 253 100 312 254 188 2880 255 207 937 256 275 913 258198 215 259 204 199 261 58.8 110 426 585 262 47.7 66.3 471 608 263 25.330.6 316 384

Additional assays that can be used to evaluate the effectiveness of atest compound include Method 4, Method 4A, Method 5 and Method 6described below:

Method 4: Aortic Ring Assay

An ex vivo assay can be used to measure the direct relaxation of a rataortic ring exposed to a test compound. As more specifically describedbelow, a test compound that inhibits PDE5 activity elicits a relaxationof the aortic ring by enhancing the cGMP signal (i.e., by inhibitingPDE5 enzyme-catalyzed hydrolysis of cGMP to GMP) evoked when the aorticring is exposed to a stable exogenous nitric oxide donor, such asdiethyltriamine NONOate (diazen-1-ium-1,2-diolate, also known as“DETA-NO”). The assay measures the EC₅₀ value for the test compound(i.e., the concentration of the test compound which produces 50% of themaximum possible effective response for the test compound).

Male Sprague-Dawley rats (250-350 g) are asphyxiated using CO₂ gas andtheir thoracic aortas carefully excised and placed in Krebs buffer. Theaortas are then carefully dissected free of connective tissue anddivided into 8 sections, each 3-4 mm in length.

Aortic rings are suspended between parallel stainless steel wires in awater jacketed (37° C.), 15 mL tissue bath under a resting tension of 1gram. Tension is measured using isometric tension transducers andrecorded using Ponemah tissue platform system. Each preparation isallowed to equilibrate for at least 60 minutes prior to compoundtesting. During this time, the tissues are also incubated with 200 uMNG-monomethyl L-arginine (“L-NMMA”), and the incubation media changedevery 15 to 20 minutes (L-NMMA is added after each wash to maintain thefinal concentration at 200 uM in each tissue bath).

Following the equilibration period, baseline tensions are recorded foreach tissue. The vasoconstrictor response to phenylepherine (1 uM) isassessed and when the response to phenylepherine reaches a maximum,vascular reactivity is subsequently assessed by a challenge ofacetylcholine (1 uM). Following another washout period, a secondbaseline value is recorded after adding the vasoconstrictornoradrenaline (25 nM) to each bath and incubating the tissues for a timeperiod (about 15 minutes) sufficient for the tissues to achieve a stabletone. An exogenous nitric oxide drive is supplied using the stablenitric oxide donor, DETA-NO. The concentration of DETA-NO is titrated(cumulatively in half-log increments) to achieve approximately 5 to 15%relaxation of the noradrenaline-evoked preconstriction. Cumulativeconcentration-response curves are constructed in a single ring,typically using 5 doses/ring and allowing 15 minutes between eachaddition.

Method 4A: Alternative Aortic Ring Assay

Method 4 can be modified to provide an alternative protocol formeasuring the direct relaxation of rat aortic rings exposed to a testcompound. This alternative method varies from Method 4 as describedbelow:

For the alternative method, the endothelium is first removed by gentlyrubbing the lumen of the vessel together between the fingers prior topreparing the rings (denuded rings). The resting tension is set at 2grams and the vasoconstrictor response to a maximal concentration ofphenylepherine (1 μM) is assessed, followed (after a washout period) bytwo further exposures to 300 nM of phenylephrine. Theconcentration-response relationship to noradrenaline is constructed ineach tissue over concentration range 0.1 to 300 nM. After anotherwashout period, the tissues are constricted with an EC₉₀ concentrationof noradrenaline for compound testing.

Method 5: Culex™ Assay

The effect of a test compound on systemic arterial blood pressure can beevaluated in a conscious pre-cannulated spontaneously hypertensive rat(“SHR”) model. This assay is conducted using an automated blood sampler(“ABS”) system. The Culex™ ABS system (Bioanalytical System, Inc., WestLafayette, Ind.) comprises a laptop computer, four control units andmetabolic cages. This ABS system allows for the collection of multipleblood samples from a single rat without causing undue stress to theanimal. In addition, the ABS system allows for the collection of urinesamples that can be potentially used for biomarker identifications.Through this approach, efficacy and standard pharmacokinetic studies areconducted in the conscious unrestrained SHR rats simultaneously todefine the relationship between plasma free drug concentration orpotential biomarker(s) and pharmacological effect (reduction of meanarterial blood pressure).

SHR rats at 12 to 16 weeks of age, weighing about 300 g, undergosurgical cannulation of both jugular veins and the right carotid artery.After surgical recovery, animals are placed in the Culex™ cages andtethered to a movement-responsive arm with a sensor that controls cagemovement when the animal moves to prevent the catheters from beingtwisted. Connections are made between the right jugular catheter and theCulex™ sterile tubing set for blood sampling, and the left jugularcatheter for compound administration, and the catheter in the rightcarotid artery is connected to a pressure transducer for monitoringblood pressure. To keep the patency of the catheters, the right jugularcannula is maintained by the “tend” function of the Culex™ that flushesthe catheter with 20 μL heparin saline (10 units/mL) every 12 minutes orbetween sampling events, and the left jugular cannula is filled withheparin saline (20 units/mL). The patency of the right carotid cannulais maintained by slow infusion of heparin saline either directly intothe extend tubing when blood pressure is not recorded or through thepressure transducer during the blood pressure monitoring. Animals areallowed to acclimate for at least two hours before compound evaluation.The test compound may be administered intravenously or by oral gavage.Blood sampling protocols (sampling time and volume) are programmed usingthe Culex™ software. The total amount of blood withdrawn from eachanimal will not exceed 750 μL/24 hrs and 10 mL/kg within two weeks.Heart rate, blood pressure, and drug concentration are monitored.Systemic arterial blood pressure and heart rate are recorded by PONEMAH(Gould Instrument System, Valley View, Ohio), a pressure transducerthrough a data acquisition system for recording blood pressure and heartrate, for 6 to 24 hours based on experimental protocol. Mean arterialblood pressure (primary endpoint) is analyzed for assessing the efficacyof the compound.

Blood samples are analyzed for measuring plasma drug concentration,using the LC/MS/MS method described below, and for evaluating potentialbiomarkers.

LC/MS/MS Method

Sample Preparation: Plasma samples (50 μL unknown, control or blank) aremixed with 10 μL acetonitrile:water or a standard solution of the testcompound and 150 μL of internal standard solution (100 ng/mL of the testcompound in acetonitrile). The mixture is centrifuged at 3000 rpm for 5min, and 125 μL of the supernatant transferred to a 96 well plate. Thesolvent is evaporated under a stream of nitrogen and the residue isreconstituted with 80 μL acetonitrile/0.1% aqueous formic acid (20:80v/v).

A 20 μL volume of each prepared sample is injected onto a PhenomenexSynergi 4 μm MAX-RP 2.0×75 mm column and eluted at 0.4 mL/min usinggradient elution from 0.1% aqueous formic acid (mobile phase A) toacetonitrile (mobile phase B). The gradient program consists of initialapplication of 90% mobile phase A, followed by a linear gradient to 75%mobile phase B from 0.2 to 1.15 min after injection and held at 75%mobile phase B until 2.0 min. The mobile phase was linearly changed backto 90% mobile phase A from 2.00 to 2.10 minutes, and the next injectiontook place at 3.00 min. Detection was performed by mass spectrometryusing positive ion electrospray (ESI) with multiple reaction monitoringof the transitions m/z 454.00 (MH+the test compound)→m/z 408.00, m/z466.24 (MH+the test compound)→409.33. The ion spray voltagea is set at5000. A calibration curve is constructed by using peak area ratios ofthe analyte relative to the internal standard. Subject concentrationsare determined by inverse prediction from their peak area ratios againstthe calibration curve.

Method 6: Implantation of Radio Transmitters and Subsequent BloodPressure Screening by Telemetry in Spontaneously Hypertensive Rats

The effect of a test compound on systemic arterial blood pressure can beevaluated in a spontaneously hypertensive rat (“SHR”) model usingtelemetry. SHR Rats are anesthetized with isoflurane gas via anisoflurane anesthesia machine that is calibrated to deliver isofluraneover a range of percentages as oxygen passes through the machine's innerchambers. The animals are placed in an induction chamber andadministered isoflurane at 4-5% to reach a surgical plane of anesthesia.They are then maintained at 1-2% during the surgical procedure via anose cone, with isoflurane delivered via a smaller isoflurane anesthesiadevice on the surgical table.

Following administration of anesthesia, the rats are implanted withtransmitters using aseptic procedures with commercially availablesterile radio-telemetry units (Data Sciences, International, Roseville,Minn. 55113-1136). Prior to surgery the surgical field is shaved,scrubbed with Dial™ brand antimicrobial solution (containing 4%chlorhexidine gluconate and 4% isopropyl alcohol) followed by anapplication of iodine (10%) spray solution. A 2.5 to 3.0 cm laparotomyis preformed and the radio-telemetry units implanted into the abdomen,with the catheter tip inserted into the abdominal aorta. Baby Weitlanerretractors are used to retain soft tissue. A 1 cm section of theabdominal aorta is partially dissected and that section cross-clampedbriefly, punctured with a 21-gauge needle and the transmitter cathetertip introduced into the vessel and secured by a single 4.0 silk sutureanchored to the adjacent psoas muscle. The transmitter body is theninserted into the abdominal cavity and simultaneously secured to theabdominal muscle wall while closing with running 4.0 silk suture. Theskin layer is closed with subdermal continuous 4.0 absorbable suture. Asubcutaneous (s.c.) administration of marcaine followed by a topicalapplication of iodine is administered into and around the suture line,respectively, upon closing. All rats receive a postoperative injectionof buprenorphine @0.05 mg/kg, s.c. before regaining consciousness. Atypical dose volume for a 0.300 kg rat will be 0.050 ml. The rats mustbe fully recovered from their operative anesthesia before theadministration of buprenorphine. They then receive the same dose oncedaily for 2 consecutive days, unless the animal demonstrates that it isin compromising postoperative pain.

Following surgery, the rats are returned to their cages and housedindividually on solid bottom caging with paper bedding. A period of noless than 7 days is allowed for recovery before experimental proceduresare initiated. It has been observed that the rats are typicallyhypertensive for several days following surgery and return to“normotensive” levels by approximately the 7^(th) day post-surgery. Theyare fed standard rat chow and water ad libitum throughout theexperimental time line.

Test compounds are administered intragastrically (i.g.) via gavage,using of a stainless steel, 2½ inch, 18 gauge gavage needle with aballed end. For single daily dosing, the target volume is 3.33 ml/kg,i.g. The dose volume for the test compound is approximately 1 ml/rat.The vehicles in which the test compound is administered ismethylcellulose (0.5%)+Tween 80 (0.1%) in 50 mM citrate buffer pH=5.0.

Blood pressure data will be obtained using Data Sciences International'sdata acquisition program (Version 3.0). Blood pressure samples arerecorded at 1.5-3 minute intervals for a 5 second duration 24 hours perday for the entire study. This data is processed by Data Science's dataanalysis software into averages of a desired time intervals. All otherdata reduction is performed in Microsoft Excel™ spreadsheets.

O. TOXICITY ASSAYS Method 7: Micronucleus Assay

An in vitro micronucleus assay can be used to determine the mutagenicpotential of a test compound. This assay detects chromosomeabnormalities resulting from exposure to a test compound by measuringthe formation of small membrane-bound DNA fragments such as micronucleiin the cytoplasm of interphase cells.

The assay is conducted using Chinese hamster ovary (CHO) cells underdifferent test conditions in the absence and presence of metabolicactivation. Concordance in outcomes between this screening assay and invitro cytogenetics is around 85%. Direct (−S9) testing is conductedusing 24 hour or 3 hour continuous treatments, whereas testing withmetabolic activation (+S9) involves a 3 hour treatment. Clastogenicityis indicated by an increase in numbers of micronucleated cells in thefirst interphase after exposure (in cytokinesis-blocked binucleatedcells). The results are compared to the level of cytotoxicity (measuredby the proportion of binucleated cells) a test compound produces. Todemonstrate validity of the test, negative (vehicle-treated) andpositive (a known responder) controls are required to respond withinestablished historical ranges.

Negative—A negative result identifies no genotoxic hazard defined by theassay's specific endpoint (ex. negative in vitro micronucleus). A testcompound displaying a negative response has not fulfilled specificevaluation criteria for the assay which may include one or both of thefollowing: 1) a reproducible concentration dependent increase in thespecific assay endpoint when compared to a negative (vehicle) controland/or 2) one or more test concentrations achieving a minimum foldincrease in the specific endpoint over controls.Positive—A positive result identifies a genotoxic hazard defined by theassay's specific endpoint (ex. positive in vitro micronucleus). A testcompound displaying a positive response has fulfilled specificevaluation criteria for the assay which may include one or both of thefollowing: 1) a reproducible concentration dependent increase in thespecific assay endpoint when compared to a negative (vehicle) controland/or 2) one or more test concentrations achieving a minimum foldincrease in the specific endpoint over controls.Equivocal—An equivocal result is reserved for situations where the testcompound has been evaluated in a valid assay test or tests (i.e. assayacceptability criteria satisfactory), but fails to display a negative orpositive result as defined by established evaluation criteria. This mayconvey the presence of a weak positive response that requires additionalrepeat testing or eventual confirmatory testing case-by-case.Inconclusive—An inconclusive result is reserved for situations where thetest compound has been evaluated in an invalid assay test (i.e. assayacceptability criteria unsatisfactory for technical reasons, ex.negative or positive controls fail to respond appropriately). Repeattesting is recommended in order to establish a valid assay test result.

The Carboxypiperidine Compound was tested in accordance with Method 7and yielded an equivocal result.

Compounds previously disclosed in examples from WO2004096810 that weretested in accordance with Method 7. The corresponding micronucleus assayresults are reported in Table G.

TABLE G WO2004096810 METHOD 7: EXAMPLE NO. MICRONUCLEUS ASSAY 261Positive 262 Equivocal 263 Positive

Additional assays that can be used to evaluate the toxicity of a testcompound (e.g., where the micronucleus assay yields an equivocal result)include Method 8, Method 9, and Method 10 described below:

Method 8: In Vitro Structural Chromosome Aberration in Human PeripheralLymphocytes

An in vitro structural chromosome aberration assay can be used toevaluate a test compound for its ability to induce structural andnumerical chromosome aberrations in the presence and absence ofmammalian metabolic activation in human peripheral lymphocytes.

Concentration calculations are based on the corresponding moiety factorof 1.000. The test compound is dissolved and diluted in DMSO, which willserve as the vehicle control at a volume equivalent to that used todeliver the test compound (1% final concentration).

Heparinized human peripheral venous blood from a healthy male volunteerare added to culture medium, followed by addition of phytohemagglutininM (Sigma Chemical Co., St. Louis, Mo.) to stimulate the cellulardivision of lymphocytes. The primary cultures are incubated for at least46 hours prior to treatment with the test compound.

The following positive control is utilized:

TABLE 1 Positive Controls Test Compound Positive Metabolic IncubationConcentration Control Activation Time (hours) (μg/mL) Cyclophosphamide(CP) + 3 10 Mitomycin C (MMC) − 3 0.4 Mitomycin C (MMC) − 24 0.1The following treatments are utilized:

TABLE 2 Treatment Conditions Test Compound Incubation Time MetabolicHarvest Time Treatment (hours) Activation (S9) (hours) (1) 3-Hr + S9 3 +24 (2) 3-Hr − S9 3 − 24 (3) 24-Hr − S9 24 − 24The following dose levels are selected for the assay:

TABLE 3 Dose Levels Treatment Concentrations (μg/mL) (1) 3-Hr + S9 100,200, 250, 300, 400, 500, 600, 700 (2) 3-Hr − S9 100, 200, 250, 300, 400,500, 600, 700 (3) 24-Hr − S9 50, 75, 100, 200, 250, 300, 350, 400Single cultures of cells for each concentration and a vehicle controlare dosed. Cell cultures are exposed to 0.1 μg/mL Colcemid® (Ciba,Switzerland) for 2 hours prior to harvest. Cells are harvested bycentrifugation, swelled in a hypotonic solution and then fixed in amethanol:glacial acetic acid fixative solution. The fixed cellsuspension is dropped onto wet microscope slides, dried, and stainedwith Giemsa. For each concentration of drug tested, at least 2 slidesper culture are prepared.

Cytotoxicity is measured by an assessment of chromosome morphology andthe inhibition of mitosis (mitotic index). A mitotic index is determinedfor all treatment conditions by scoring 1000 cells per condition for theproportion of metaphase cells.

The maximum concentration selected for scoring chromosome aberrations isthe highest dose at which a sufficient number of analyzable metaphasecells are expected. If possible, the highest dose selected wouldsuppress the mitotic index by approximately 50%, but not greater than70% reduction.

At least one concentration under each test condition is selected foranalysis along with the concurrent vehicle and positive control. Theslides are not analyzed in a blinded fashion. At the discretion of thestudy director, additional test concentrations may be evaluated afterthe initial evaluation has been completed to provide furtherclarification of an effect (i.e., an increase or absence ofaberrations).

The highest test concentration selected for analysis should meet 1 ofthe following criteria: (1) produces approximately a 50% reduction ofthe mitotic index compared to the vehicle controls, (2) shows evidenceof incomplete solubility, or (3) is equivalent to the maximumconcentration of 5000 μg/mL or 10 mM (whichever is lower). Whenpossible, 100 acceptable diploid metaphase cells from each culture areevaluated for chromosome damage. An exception to this is an apparentincrease in the frequency of abnormal cells during data collection(i.e., >10 aberrant cells are tallied in the first 50 acceptable cells)in which case further analysis is discontinued. Metaphase cells selectedfor analysis must be intact (i.e., have 46±2 chromosomes), and have aminimum of overlapping chromosomes. In addition, the chromosomes shouldappear elongated such that the individual arms and centromeric regionscan be readily identified. Each metaphase cell is classified as normalor abnormal with the type(s) and number of abnormalities present percell recorded. For abnormal cells, the stage X and Y vernierco-ordinates of the slide are recorded. Structural chromosomeaberrations are classified as chromatid break (Ct brk), chromatidfragments (Ct Frg), chromatid damage including exchanges, rings,dicentrics and translocations (R), chromosome breaks (CsBrk), chromosomefragments (Cs Frg) and multiple breaks (M). In addition, cells withpulverized chromosomes (PV) are collected in the total aberration tally.Cells that contained gaps are recorded but not included in the totalaberration tally. Numerical chromosome aberrations are determined foreach treatment condition and the concurrent vehicle controls byevaluating the number of polyploid and endoreduplicated cells. Thepolyploid indices are obtained by scoring when possible 1000 metaphasecells/culture while tabulating the number of metaphase cells that arepolyploid or endoreduplicated.

A Fisher's Exact 1-tailed test on the total number of aberrant cellsfrom each treatment group compared to the total aberrant cells from thenegative controls are used as the statistical analysis.⁵ A p-value≦0.05is considered to be statistically significant.An assay is considered valid if the following criteria are met:

-   -   1. Positive control induces a statistically significant increase        in the percentage of cells with chromosome aberrations when        compared to the concurrent vehicle control and the induced        frequencies are comparable to published or historical data.    -   2. Vehicle control cultures have ≦3% of cells with aberrations        or a percentage considered comparable to published or historical        data.    -   3. Approximately 50% inhibition of mitotic index is observed at        the highest dose level. This requirement is not applicable to        test compounds for which no apparent cytotoxicity could be        achieved at the maximum soluble concentration or highest        allowable dose.

Method 9: Seven Day Oral Gavage Toxicity Study in the Male SD/IGS Rat

The toxicity of a test compound can be evaluated in a rat model. Thisstudy determines the potential toxicity of and systemic exposure to testcompounds, when administered by oral gavage, once daily, for 7consecutive days, to 6 to 8 week old male Charles River SD/IGS ratsweighing between 175 g and 200 g. This study is conducted using theXybion Path/Tox System (Xybion Medical Systems Corporation, CedarKnolls, N.J.).

The test compound is dosed orally once for 7 days by gavage at a volumeof 10 mL/kg of body weight. The test compound is administered to 3groups of rats (n=5) getting doses of 30 mg/kg/day, 100 mg/kg/day, and500 mg/kg/day, respectively. A control group (n=5) receives vehicle(0.5% Methylcellulose (w/v) and 0.1% Polysorbate 80 (v/v) in 50 mMphosphate buffer). The oral route is used because it is the intendedroute of exposure in humans. The appropriate amount of test compound issuspended in 0.5% methylcellulose (w/v) and 0.1% polysorbate 80 (v/v) in50 mM citrate buffer. The dosing suspension pH is maintained between 3and 9.

Every animal is serially bled at 1, 3, 8, and 24 hours post dose onDay 1. Urine is collected approximately 24 hours for metabolic analysis.Survival and moribundity observations are conducted once daily duringthe pre treatment period. Clinical signs are observed daily, 1-3 hourspost dose. Animals are euthanized via exsanguination after 7 days.Animals found dead are refrigerated and necropsied at the earliestpossible time (within working hours). Terminal body weights, bloodsamples and organ weights are not taken from animals found dead.Moribund/unscheduled/scheduled animals that are sacrificed are takenimmediately to necropsy, and body weights, blood samples, and allclinical pathology samples except for urine samples are taken.

Various tissues (adrenal, femur bone, brain, cecum, colon, duodenum,epididymis, heart, ileum, jejunum, kidney, liver, pancreas, bicepsfemoris skeletal muscle, spleen, stomach, testis, thymus, thyroid,lumbar spinal cord, and mesenteric lymph node) will be weighed, flashfreezed stained and fixed. After macroscopic examination, tissuescollected with gross abnormalities are retained in formalin. All tissuescollected with gross abnormalities are processed and examinedmicroscopically by the pathologist. Tissues are trimmed, embedded,sectioned, and stained with hematoxylin and eosin and examinedmicroscopically by the pathologist at the pathologist's discretion. Bonemarrow smears are prepared and stained with Wright's stain. To elucidatethe nature of an individual animal's tissue change, additional tissuecollection, sectioning, staining, and microscopic examination isconducted as requested by the pathologist.

Clinical observations, body weights, gross necropsy observations, andhistopathologic findings are directly entered into the Xybion Path/ToxSystem.

Method 10: BioLum Ames Gene Mutation Assay

A Salmonella mutagenicity test (also known as the Ames test) can be usedto determine the mutagenic potential of a test compound. This assaymeasures the mutation rate of bacteria that are exposed to a testcompound. See, for example, Ames, B. N., Durston, W. E., Yamasaki, E.and Lee, F. D. (1973) Carcinogens are mutagens: a simple test systemcombining liver homogenates for activation and bacteria for detection.Proc. Natl. Acad. Sci. USA, 70, 2281-2285). Some carcinogens becomeactivated when they are enzymatically transformed to an electrophilicspecies capable of covalently binding to DNA. In the Ames assay, S9(9000 g supernatant) fractions are prepared from the livers of ratspretreated with phenobarbital (PB)/5,6-benzoflavone (BF) or Aroclor 1254to induce such drug metabolizing enzyme activity.

The BioLum Ames assay is a higher throughput screening version of thestandard bacterial (Ames) gene mutation assay. To demonstrate validityof the test, negative (vehicle-treated) and positive (a known responder)controls are required to respond within established historical ranges.

Negative—A negative result identifies no genotoxic hazard defined by theassay's specific endpoint (ex. negative in vitro micronucleus). A testcompound displaying a negative response has not fulfilled specificevaluation criteria for the assay which may include one or both of thefollowing: 1) a reproducible concentration dependent increase in thespecific assay endpoint when compared to a negative (vehicle) controland/or 2) one or more test concentrations achieving a minimum foldincrease in the specific endpoint over controls.Positive—A positive result identifies a genotoxic hazard defined by theassay's specific endpoint (ex. positive in vitro micronucleus). A testcompound displaying a positive response has fulfilled specificevaluation criteria for the assay which may include one or both of thefollowing: 1) a reproducible concentration dependent increase in thespecific assay endpoint when compared to a negative (vehicle) controland/or 2) one or more test concentrations achieving a minimum foldincrease in the specific endpoint over controls.Equivocal—An equivocal result is reserved for situations where the testcompound has been evaluated in a valid assay test or tests (i.e. assayacceptability criteria satisfactory), but fails to display a negative orpositive result as defined by established evaluation criteria. This mayconvey the presence of a weak positive response that requires additionalrepeat testing or eventual confirmatory testing case-by-case.Inconclusive—An inconclusive result is reserved for situations where thetest compound has been evaluated in an invalid assay test (i.e. assayacceptability criteria unsatisfactory for technical reasons, ex.negative or positive controls fail to respond appropriately). Repeattesting is recommended in order to establish a valid assay test result.

P. PHARMACOKINETIC/PHARMACODYNAMIC STUDY

Method 11: Single Dose Pharmacokinetics and Oral Bioavailability in MaleSprague-Dawley Rat Following Intravenous and Oral Administration

An in vivo model can be used to evaluate the single dose pharmacokineticproperties and absolute oral bioavailability of a test compound. Asdescribed more specifically below, a test compound is administered toSprague-Dawley (SD) rats either intravenously or orally in a crossoverstudy design and the resulting pharmacokinetic properties and oralbioavailability are measured.

Male rats are administered a 2.0 mg/kg dose orally (n=2) by gavage as asuspension (0.5% methylcellulose/0.1% Tween 80 in distilled water).After a 72 hour wash-out period, the same rats were administered 2.0mg/kg dose bolus intravenously (n=2) as a solution (70% PEG 400/20% 0.05M Citrate Buffer pH 3/10% Ethanol). Serial blood samples (for plasma)are collected from each rat over a 24 hour post dose for each route.Plasma concentrations of test compound are determined using a LC/MS/MSmethod with a lower limit of quantitation (LLOQ) of 1.2 (for −534)ng/mL. Pharmacokinetic parameters of a test compound are determined fromthe plasma concentration-time data using non-compartmental methods.

LC/MS/MS: 1) Column: Hyperso; AQUASIL C-18 2.1×20 mm, 3.0 μm; 2) Mobilephase: Aqueous Water with 0.1% Formic Acid, Organic: Acetonitrile;Ionization: +ESI (API 4000). MRM: m/z 494.4→m/z 394.0 (Example 261 inWO2004096810), m/z 509.44→m/z 409.80 (Example 263 in WO2004096810), m/z495.33→m/z 395.20 (Example 262 in WO2004096810). The detection limitsare 0.12 ng/mL (Example 261 in WO2004096810), 1.3 ng/mL (Example 263 inWO2004096810) and 0.11 ng/mL (Example 262 in WO2004096810).

Watson (Version 6.4.0.04) is used to calculate mean test compoundconcentrations, corresponding standard deviations (SD), and percentcoefficient of variation (% CV), and to estimate pharmacokineticparameters (derived by noncompartmental methods) and associatedstatistics (mean, SD & CV %) if applicable. (Since n=2, no SD or CVcalculated?). Concentrations below the limit of quantitation (BLQ) arereported as zero (0) and are used in the evaluation of meanconcentrations and the estimation of AUC. The peak plasma concentration(C_(max)) and the time to reach peak concentration (tmax) are recordeddirectly from individual plasma concentration-time profiles. Theterminal log-linear phase of the plasma concentration-time curve isidentified by linear regression of data points. The terminal half-life(t_(1/2)) is calculated as ln(2) divided by absolute value of the slopeof the terminal log-linear phase. The area under the plasmaconcentration-time curve from time zero to time of the last quantifiableconcentration (t) [AUC_((0-t))] is determined using the lineartrapezoidal method. The area under the plasma concentration-time curvefrom time zero to infinity [AUC_((0-∞))] is determined as AUC_((0-t))plus the extrapolated area. The extrapolated area is determined bydividing the last observed plasma concentration by the slope of theterminal log-linear phase. Systemic plasma clearance (CL) is calculatedas dose/AUC_((0-∞)) while the volume of distribution at steady state(V_(dss)) is calculated as CL×MRT, where MRT (mean residence time) isdefined as AUMC_((0-∞))/AUC_((0-∞)). The absolute PO bioavailability (F)is calculated as a ratio of the dose-normalized AUC_((0-∞)) ofindividual animal after PO administration to the dose-normalizedAUC_((0-∞)) of individual animal after IV administration given thecrossover study design. The peak plasma concentration (C_(max)), thetime to reach peak concentration (t_(max)), the terminal half-life(t_(1/2)), the area under the plasma concentration-time curve from timezero to infinity [AUC_((0-∞))], the volume of distribution at steadystate (V_(dss)), the systemic plasma clearance (CL), and the absolute PObioavailability (F) are shown in Table C.

The Carboxypiperidine Compound was tested in accordance with Method 11and the results are reported in Table H. Compounds previously disclosedin examples from WO2004096810 were tested in accordance with Method 11.Results for these compounds are also are reported in Table H.

TABLE H Summary of Mean PK Parameters Dose C_(max) t_(max) t_(1/2) AUC(0-∞) V_(dss) CL F Compound Route (mg/kg) N (ng/mL) (hr) (hr) (ng ·hr/mL) (L/kg) (mL/min/kg) (%) Carboxypiperidine iv 2 2 6.3 17500 0.7151.9 Compound po 2 3 1360 ± 216 0.5 ± 0.2 5.7 ± 0.2 9070 ± 2300 51.8 ±13.1 WO2004096810, iv 2 2 6.4 19017 0.564 1.75 Example 261 po 2 2 23510.67 6.7 20040 105 WO2004096810, iv 2 2 5.5 440 6.11 76.3 Example 262 po2 2 87.1 0.67 3.4 280 63.9 WO2004096810, iv 2 2 4.5 555 5.07 60.1Example 263 po 2 2 102 0.33 3.8 329 60.0

Q. BIOLOGICAL PROTOCOLS Coadministration with Angiotensin ConvertingEnzyme Inhibitor Method 12: SHR Rat Combination Therapy

A study was conducted to assess the effect of repeated oral dosing ofthe Carboxypiperidine Compound on blood pressure lowering and whetherco-administration of the Carboxypiperidine Compound and the angiotensinconverting enzyme inhibitor enalapril could result in further bloodpressure lowering. The study consisted of treating spontaneouslyhypertensive rats (SHR) telemetered as described in Method F with theCarboxypiperidine Compound (1 mg/kg once daily oral) and enalapril(0.007% in drinking water) alone and in combination for 7 days (FIG. 1)(n=12/group). The Carboxypiperidine Compound reduced blood pressure froma pre-dose baseline value in comparison to a vehicle treated group by11±1 mmHg on day 1. The reduction in blood pressure was sustained forthe 7-day period, remaining 9±1 mmHg below the pre-dose baseline on day7.

Enalapril also reduced blood pressure from a pre-dose baseline value incomparison to a vehicle treated group with a maximal reduction of 22±2mmHg on day 7. The combination of the Carboxypiperidine Compound plusenalapril reduced blood pressure more than blood pressure was reduced byeach agent alone on all days.

Changes in the urinary cGMP mechanistic biomarker were assessed over the24-hour period on day 9. cGMP was elevated in a 0-24 hour urinecollection in both the Carboxypiperidine Compound and theCarboxypiperidine Compound+enalapril combination groups in comparison tothe vehicle treated group (FIG. 2) (n=12/group).

All documents mentioned in this application are expressly incorporatedby reference as if fully set forth at length. When introducing elementsof the present invention or the preferred embodiment(s) thereof, thearticles “a”, “an”, “the” and “said” are intended to mean that there areone or more of the elements. The terms “comprising”, “including” and“having” are intended to be inclusive and mean that there may beadditional elements other than the listed elements.

1. A compound, and pharmaceutically acceptable salts of the compound,having the structure:


2. The compound of claim 1 that is a free acid.
 3. The pharmaceuticallyacceptable salts of the compound of claim
 1. 4. A pharmaceuticalcomposition comprising a compound, or a pharmaceutically acceptable saltof the compound, having the structure:

a pharmaceutically-acceptable carrier.
 5. The pharmaceutical compositionof claim 4 comprising a free acid of the compound.
 6. The pharmaceuticalcomposition of claim 4 comprising a pharmaceutically acceptable salt ofthe compound.
 7. The pharmaceutical composition of claim 4 furthercomprising an angiotensin converting enzyme inhibitor.
 8. Thepharmaceutical composition of claim 4 further comprising an angiotensinII receptor antagonist.
 9. A method of treating a condition in asubject, the method comprising administering to the subject atherapeutically-effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, having the structure:

wherein the condition is selected from the group consisting of acardiovascular condition, a metabolic condition, a central nervoussystem condition, a pulmonary condition, sexual dysfunction, pain andrenal dysfunction.
 10. The method of claim 9 wherein the condition is acardiovascular condition.
 11. The method of claim 10 wherein thecardiovascular condition is hypertension.
 12. The method of claim 10wherein the cardiovascular condition is heart failure.
 13. The method ofclaim 10 wherein the cardiovascular condition is angina.
 14. The methodof claim 9 further comprising administering a therapeutically-effectiveamount of an angiotensin converting enzyme inhibitor to the subject. 15.The method of claim 9 further comprising administering atherapeutically-effective amount of an angiotensin II receptorantagonist to the subject.
 16. Use of a compound, or a pharmaceuticallyacceptable salt thereof, having the structure:

in the manufacture of a medicament for the treatment of a conditionselected from the group consisting of cardiovascular conditions,metabolic conditions, central nervous system conditions, pulmonaryconditions, sexual dysfunction, pain and renal dysfunction.
 17. The useof claim 16 wherein the condition is a cardiovascular condition.
 18. Theuse of claim 17 wherein the cardiovascular condition is hypertension.19. The use of claim 17 wherein the cardiovascular condition is heartfailure.
 20. The use of claim 18 wherein the cardiovascular condition isangina.
 21. A method of preparing a first compound having the structure:

The method comprising contacting a second compound having the structure:

with isonipecotic acid to provide the first compound.